AC/DC voltage apparatus for detection of nucleic acids

ABSTRACT

The invention relates to nucleic acids covalently coupled to electrodes via conductive oligomers. More particularly, the invention is directed to the site-selective modification of nucleic acids with electron transfer moieties and electrodes to produce a new class of biomaterials, and to methods of making and using them.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuing application of provisional application Ser. No.60/040,155 filed 7 Mar. 1997.

FIELD OF THE INVENTION

The invention relates to nucleic acids covalently coupled to electrodesvia conductive oligomers. More particularly, the invention is directedto the site-selective modification of nucleic acids with electrontransfer moieties and electrodes to produce a new class of biomaterials,and to methods of making and using them.

BACKGROUND OF THE INVENTION

The detection of specific nucleic acids is an important tool fordiagnostic medicine and molecular biology research. Gene probe assayscurrently play roles in identifying infectious organisms such asbacteria and viruses, in probing the expression of normal genes andidentifying mutant genes such as oncogenes, in typing tissue forcompatibility preceding tissue transplantation, in matching tissue orblood samples for forensic medicine, and for exploring homology amonggenes from different species.

Ideally, a gene probe assay should be sensitive, specific and easilyautomatable (for a review, see Nickerson, Current Opinion inBiotechnology 4:48-51 (1993)). The requirement for sensitivity (i.e. lowdetection limits) has been greatly alleviated by the development of thepolymerase chain reaction (PCR) and other amplification technologieswhich allow researchers to amplify exponentially a specific nucleic acidsequence before analysis (for a review, see Abramson et al., CurrentOpinion in Biotechnology, 4:41-47 (1993)).

Specificity, in contrast, remains a problem in many currently availablegene probe assays. The extent of molecular complementarity between probeand target defines the specificity of the interaction. Variations in theconcentrations of probes, of targets and of salts in the hybridizationmedium, in the reaction temperature, and in the length of the probe mayalter or influence the specificity of the probe/target interaction.

It may be possible under some limited circumstances to distinguishtargets with perfect complementarity from targets with mismatches,although this is generally very difficult using traditional technology,since small variations in the reaction conditions will alter thehybridization. New experimental techniques for mismatch detection withstandard probes include DNA ligation assays where single pointmismatches prevent ligation and probe digestion assays in whichmismatches create sites for probe cleavage.

Finally, the automation of gene probe assays remains an area in whichcurrent technologies are lacking. Such assays generally rely on thehybridization of a labelled probe to a target sequence followed by theseparation of the unhybridized free probe. This separation is generallyachieved by gel electrophoresis or solid phase capture and washing ofthe target DNA, and is generally quite difficult to automate easily.

The time consuming nature of these separation steps has led to twodistinct avenues of development. One involves the development ofhigh-speed, high-throughput automatable electrophoretic and otherseparation techniques. The other involves the development ofnon-separation homogeneous gene probe assays.

PCT application WO 95/15971 describes novel compositions comprisingnucleic acids containing electron transfer moieties, includingelectrodes, which allow for novel detection methods of nucleic acidhybridization.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the invention to provide for improvedmethods and compositions of nucleic acids covalently attached toelectrodes and at least one other electron transfer moiety.

In one aspect, the present invention provides methods for detecting thepresence of a target sequence in a nucleic acid sample. The methodcomprises applying a first input signal comprising an AC component and anon-zero DC component to a hybridization complex comprising at least atarget sequence and a first probe single stranded nucleic acid. Thehybridization complex is covalently attached to a first electrontransfer moiety comprising an electrode, and a second electron transfermoiety. The presence of the hybridization complex is detected byreceiving an output signal characteristic of electron transfer throughsaid hybridization complex.

In an alternative aspect, the invention provides methods as abovewherein a first input signal comprising an AC component at a firstfrequency and a non-zero DC component is applied to a hybridizationcomplex, and then a second input signal comprising an AC component atleast a second frequency and a non-zero DC component is applied, suchthat the presence of the hybridization complex can be detected.

In a further aspect of the invention, the methods utilize a first inputsignal comprising an AC component and a first non-zero DC componentapplied to a hybridization complex, followed by a second input signalcomprising said AC component and at least a second non-zero DCcomponent.

In an additional aspect, the invention provides methods that utilize afirst input signal comprising an AC component at a first voltageamplitude, and a second input signal comprising said AC component at asecond voltage amplitude applied to the hybridization complex.

In an additional aspect, the invention provides methods utilizinghybridization complex comprising a single stranded nucleic acidcovalently attached to both a first electron transfer moiety comprisingan electrode, and a second electron transfer moiety, and a targetsequence hybridized to said single stranded nucleic acid.

In a further aspect, the hybridization complexes comprise a singlestranded nucleic acid covalently attached via a conductive oligomer to afirst electron transfer moiety comprising an electrode, and a targetsequence hybridized to said single stranded nucleic acid, and a secondelectron transfer moiety.

In a further aspect, the invention provides apparatus for the detectionof target nucleic acids in a test sample, comprising a test chambercomprising a first and a second measuring electrode, wherein the firstmeasuring electrode comprises a covalently attached conductive oligomercovalently attached to a single stranded nucleic acid, and an AC/DCvoltage source electrically connected to the test chamber.

In an additional aspect, the invention provides apparatus comprising atest chamber comprising a first and a second measuring electrode,wherein the first measuring electrode comprises a covalently attachedsingle stranded nucleic acid comprising a covalently attached secondelectron transfer moiety, and an AC/DC voltage source electricallyconnected to the test chamber.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the synthetic scheme for a conductive oligomer covalentlyattached to a uridine nucleoside via an amide bond.

FIG. 2 depicts the synthetic scheme for covalently attaching aconductive oligomer covalently attached to a uridine nucleoside via anamine bond.

FIG. 3 depicts the synthetic scheme for a conductive oligomer covalentlyattached to a uridine nucleoside via the base.

FIG. 4 depicts the synthetic scheme for a conductive oligomer covalentlyattached to a nucleoside via a phosphate of the ribose-phosphatebackbone. The conductive oligomer is a phenyl-acetylene Structure 5oligomer, although other oligomers may be used, and terminates in anethyl pyridine protecting group, as described herein, for attachment togold electrodes.

FIG. 5 depicts the synthetic scheme for a conductive oligomer covalentlyattached to a nucleoside via a phosphate of the ribose-phosphatebackbone, using an amide linkage and an ethylene linker, although otherlinkers may be used. The conductive oligomer is a phenyl-acetyleneStructure 5 oligomer, although other oligomers may be used, andterminates in an ethyl pyridine protecting group, as described herein,for attachment to gold electrodes.

FIG. 6 depicts the synthetic scheme for a conductive polymer containingan aromatic group with a substitution group. The conductive oligomer isa phenyl-acetylene Structure 5 oligomer with a single methyl R group oneach phenyl ring, although other oligomers may be used, and terminatesin an ethyl pyridine protecting group, as described herein, forattachment to gold electrodes.

FIG. 7 depicts the synthetic scheme for the synthesis of a metallocene,in this case ferrocene, linked via a conductive oligomer to anelectrode. The conductive oligomer is a phenyl-acetylene Structure 5oligomer, although other oligomers may be used, and terminates in anethyl pyridine protecting group, as described herein, for attachment togold electrodes.

FIG. 8 depicts a model compound, ferrocene attached to a C₁₆ alkanemolecule (insulator-1), at 200 mV AC amplitude and frequencies of 1, 5and 100 Hz. The sample responds at all three frequencies, with highercurrents resulting from higher frequencies.

FIGS. 9A and 9B depict the response with varying frequency. FIG. 9Ashows overlaid voltammograms of an electrode coated with aferrocene-conductive oligomer model complex (wire-2). Four excitationfrequencies were applied, 10 Hz, 100 Hz, 1 kHz and 10 kHz, all at 25 mVoverpotential. Again, current increases with frequency. FIG. 9B showsoverlaid voltammograms of electrodes coated with either ssDNA or dsDNA.ssDNA was run at 1 Hz and 10 Hz at 100 mV overpotential (bottom twolines). dsDNA was run at 1, 10, 50 and 100 Hz at 10 mV overpotential(top four lines). Note that the scales between FIG. 8 and FIGS. 9A and9B are different.

FIG. 10 depicts the frequency response of these systems. The peakcurrents at a number of frequencies are determined and plotted. Sample 3(filled triangles) responds to increasing frequencies through 10 kHz(system limit), while samples 1 (open circles) and 2 (filled circles)lose their responses at between 20 and 200 Hz. This data was notnormalized to the increase in current associated with increasingfrequency.

FIG. 11 depicts the frequency responses of ssDNA (open circles; sample5) and dsDNA (filled circles; sample 6) at 25 mV overpotential. Thecurrent has been normalized. The curves are not a fit to the data;rather, these are models of RC circuits, illustrating that the data canbe fit to such curves, and that the system is in fact mimic standard RCcircuits. The top curve was modeled using a 500 ohm resistor and a 0.001farad capacitor. The bottom curve was modeled using a 20 ohm resistorand a 0.002 farad capacitor.

FIG. 12 shows that increasing the overpotential will increase the outputcurrent.

FIGS. 13A and 13B illustrate that the overpotential and frequency can betuned to increase the selectivity and sensitivity, using Sample 1.

FIG. 14 shows that ferrocene added to the solution (Sample 7; opencircles) has a frequency response related to diffusion that is easilydistinguishable from attached ferrocene (Sample 3; filled circles).

FIGS. 15A and 15B shows the phase shift that results with differentsamples. FIG. 15A uses two experiments of Sample 1, Sample 3 and Sample4. FIG. 15B uses Sample 5 and Sample 6.

FIG. 16 depicts the synthetic scheme for a conductive oligomercovalently attached to a uridine nucleoside via an amine bond, with aCH2 group as a Z linker. Compound C4 can be extended as outlined hereinand in FIG. 1.

FIGS. 17A, 17B, 17C, 17D, 17E, 17F and 17G depict other conductiveoligomers, attached either through the base (A-D) or through the riboseof the backbone (E-G), which have been synthesized using the techniquesoutlined herein. FIG. 17H depicts a conductive oligomer attached to aferrocene. As will be appreciated by those in the art, the compounds areshown as containing CPG groups, phosphoramidite groups, or neither;however, they may all be made as any of these.

FIG. 18 depicts a synthetic scheme for a four unit conductive oligomerattached to the base.

FIG. 19 depicts a synthetic scheme for a four unit conductive oligomerattached to the base.

FIG. 20 depicts the use of a trimethylsilylethyl protecting group insynthesizing a five unit wire attached via the base.

FIG. 21 depicts the use of a trimethylsilylethyl protecting group insynthesizing a five unit wire attached via the base.

FIGS. 22A and 22B depict simulations based on traditionalelectrochemical theory (FIG. 22B) and the simulation model developedherein (FIG. 22A).

FIGS. 23A and 23B depict experimental data plotted with theoreticalmodel, showing good correlation. Fc-wire of Example 7 was used as 10 Hz(FIG. 23A) and 100 Hz (FIG. 23B).

DETAILED DESCRIPTION OF THE INVENTION

The present invention capitalizes on the previous discovery thatelectron transfer apparently proceeds through the stacked π-orbitals ofthe heterocyclic bases of double stranded (hybridized) nucleic acid(“the π-way”). This finding allows the use of nucleic acids containingelectron transfer moieties to be used as nucleic acid probes. See PCTpublication WO 95/15971, hereby incorporated by reference in itsentirety, and cited references. This publication describes thesite-selective modification of nucleic acids with redox active moieties,i.e. electron donor and acceptor moieties, which allow the long-distanceelectron transfer through a double stranded nucleic acid. In general,electron transfer between electron donors and acceptors does not occurat an appreciable rate when the nucleic acid is single stranded, nordoes it occur appreciably unless nucleotide base pairing exists in thedouble stranded sequence between the electron donor and acceptor in thedouble helical structure. Thus, PCT publication WO 95/15971 and thepresent invention are directed to the use of nucleic acids with electrontransfer moieties, including electrodes, as probes for the detection oftarget sequences within a sample.

In one embodiment, the present invention provides for novel gene probes,which are useful in molecular biology and diagnostic medicine. In thisembodiment, single stranded nucleic acids having a predeterminedsequence and covalently attached electron transfer moieties, includingan electrode, are synthesized. The sequence is selected based upon aknown target sequence, such that if hybridization to a complementarytarget sequence occurs in the region between the electron donor and theelectron acceptor, electron transfer proceeds at an appreciable anddetectable rate. Thus, the invention has broad general use, as a newform of labelled gene probe. In addition, the probes of the presentinvention allow detection of target sequences without the removal ofunhybridized probe. Thus, the invention is uniquely suited to automatedgene probe assays or field testing.

The present invention provides improved compositions comprising nucleicacids covalently attached via conductive oligomers to an electrode, of ageneral structure depicted below in Structure 1:

In Structure 1, the hatched marks on the left represent an electrode. Xis a conductive oligomer as defined herein. F₁ is a linkage that allowsthe covalent attachment of the electrode and the conductive oligomer,including bonds, atoms or linkers such as is described herein, forexample as “A”, defined below. F₂ is a linkage that allows the covalentattachment of the conductive oligomer to the nucleic acid, and may be abond, an atom or a linkage as is herein described. F₂ may be part of theconductive oligomer, part of the nucleic acid, or exogeneous to both,for example, as defined herein for “Z”.

By “nucleic acid” or “oligonucleotide” or grammatical equivalents hereinmeans at least two nucleotides covalently linked together. A nucleicacid of the present invention will generally contain phosphodiesterbonds, although in some cases, as outlined below, nucleic acid analogsare included that may have alternate backbones, comprising, for example,phosphoramide (Beaucage et al., Tetrahedron 49(10):1925 (1993) andreferences therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl etal., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res.14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al.,J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta26:141 91986)), phosphorothioate, phosphorodithioate,O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides andAnalogues: A Practical Approach, Oxford University Press), and peptidenucleic acid backbones and linkages (see Egholm, J. Am. Chem. Soc.114:1895 (1992); Meier et al., Chem. Int. Ed. Engl. 31:1008 (1992);Nielsen, Nature, 365:566 (1993); Carlsson et al., Nature 380:207 (1996),all of which are incorporated by reference). Nucleic acids containingone or more carbocyclic sugars are also included within the definitionof nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995) pp169-176). Several nucleic acid analogs are described in Rawls, C & ENews Jun. 2, 1997 page 35, hereby incorporated by reference. Thesemodifications of the ribose-phosphate backbone may be done to facilitatethe addition of electron transfer moieties, or to increase the stabilityand half-life of such molecules in physiological environments.

Particularly preferred are peptide nucleic acids (PNA). These backbonesare substantially non-ionic under neutral conditions, in contrast to thehighly charged phosphodiester backbone of naturally occurring nucleicacids. This results in two advantages. First, the PNA backbone exhibitsimproved hybridization kinetics. PNAs have larger changes in the meltingtemperature (Tm) for mismatched versus perfectly matched basepairs. DNAand RNA typically exhibit a 2-4° C. drop in Tm for an internal mismatch.With the non-ionic PNA backbone, the drop is closer to 7-9° C. Thisallows for better detection of mismatches. Similarly, due to theirnon-ionic nature, hybridization of the bases attached to these backbonesis relatively insensitive to salt concentration. This is particularlyadvantageous in the systems of the present invention, as a reduced salthybridization solution has a lower Faradaic current than a physiologicalsalt solution (in the range of 150 mM).

The nucleic acids may be single stranded or double stranded, asspecified, or contain portions of both double stranded or singlestranded sequence. The nucleic acid may be DNA, both genomic and cDNA,RNA or a hybrid, where the nucleic acid contains any combination ofdeoxyribo- and ribo-nucleotides, and any combination of bases, includinguracil, adenine, thymine, cytosine, guanine, inosine, xathanine andhypoxathanine, etc. As used herein, the term “nucleoside” includesnucleotides, and modified nucleosides such as amino modifiednucleosides.

The nucleosides and nucleic acids are covalently attached to aconductive oligomer. By “conductive oligomer” herein is meant asubstantially conducting oligomer, preferably linear, some embodimentsof which are referred to in the literature as “molecular wires”. By“substantially conducting” herein is meant that the rate of electrontransfer through the conductive oligomer is faster than the rate ofelectron transfer through single stranded nucleic acid, such that theconductive oligomer is not the rate limiting step in the detection ofhybridization, although as noted below, systems which use spacers thatare the rate limiting step are also acceptable. Stated differently, theresistance of the conductive oligomer is less than that of the nucleicacid. Preferably, the rate of electron transfer through the conductiveoligomer is faster than the rate of electron transfer through doublestranded nucleic acid, i.e. through the stacked π-orbitals of the doublehelix. Generally, the conductive oligomer has substantially overlappingπ-orbitals, i.e. conjugated π-orbitals, as between the monomeric unitsof the conductive oligomer, although the conductive oligomer may alsocontain one or more sigma (σ) bonds. Additionally, a conductive oligomermay be defined functionally by its ability to inject or receiveelectrons into or from an attached nucleic acid. Furthermore, theconductive oligomer is more conductive than the insulators as definedherein.

In a preferred embodiment, the conductive oligomers have a conductivity,S, of from between about 10⁻⁶ to about 10⁴ Ω⁻¹cm⁻¹, with from about 10⁻⁵to about 10³ Ω⁻¹cm⁻¹ being preferred, with these S values beingcalculated for molecules ranging from about 20 Å to about 200 Å. Asdescribed below, insulators have a conductivity S of about 10⁻⁷ Ω⁻¹cm⁻¹or lower, with less than about 10⁻⁸ Ω⁻¹cm⁻¹ being preferred. Seegenerally Gardner et al., Sensors and Actuators A 51 (1995) 57-66,incorporated herein by reference.

Desired characteristics of a conductive oligomer include highconductivity, sufficient solubility in organic solvents and/or water forsynthesis and use of the compositions of the invention, and preferablychemical resistance to reactions that occur i) during nucleic acidsynthesis (such that nucleosides containing the conductive oligomers maybe added to a nucleic acid synthesizer during the synthesis of thecompositions of the invention), ii) during the attachment of theconductive oligomer to an electrode, or iii) during hybridizationassays.

The oligomers of the invention comprise at least two monomeric subunits,as described herein. As is described more fully below, oligomers includehomo- and hetero-oligomers, and include polymers.

In a preferred embodiment, the conductive oligomer has the structuredepicted in Structure 2:

As will be understood by those in the art, all of the structuresdepicted herein may have additional atoms or structures; i.e. theconductive oligomer of Structure 2 may be attached to electron transfermoieties, such as electrodes, transition metal complexes, organicelectron transfer moieties, and metallocenes, and to nucleic acids, orto several of these. Unless otherwise noted, the conductive oligomersdepicted herein will be attached at the left side to an electrode; thatis, as depicted in Structure 2, the left “Y” is connected to theelectrode as described herein and the right “Y”, if present, is attachedto the nucleic acid, either directly or through the use of a linker, asis described herein.

In this embodiment, Y is an aromatic group, n is an integer from 1 to50, g is either 1 or zero, e is an integer from zero to 10, and m iszero or 1. When g is 1, B—D is a conjugated bond, preferably selectedfrom acetylene, alkene, substituted alkene, amide, azo, —C═N— (including—N═C—, —CR═N— and —N═CR—), —Si═Si—, and —Si═C— (including —C═Si—,—Si═CR— and —CR═Si—). When g is zero, e is preferably 1, D is preferablycarbonyl, or a heteroatom moiety, wherein the heteroatom is selectedfrom oxygen, sulfur, nitrogen or phosphorus. Thus, suitable heteroatommoieties include, but are not limited to, —NH and —NR, wherein R is asdefined herein; substituted sulfur; sulfonyl (—SO₂—) sulfoxide (—SO—);phosphine oxide (—PO— and —RPO—); and thiophosphine (—PS— and —RPS—).However, when the conductive oligomer is to be attached to a goldelectrode, as outlined below, sulfur derivatives are not preferred.

By “aromatic group” or grammatical equivalents herein is meant anaromatic monocyclic or polycyclic hydrocarbon moiety generallycontaining 5 to 14 carbon atoms (although larger polycyclic ringsstructures may be made) and any carbocylic ketone or thioketonederivative thereof, wherein the carbon atom with the free valence is amember of an aromatic ring. Aromatic groups include arylene groups andaromatic groups with more than two atoms removed. For the purposes ofthis application aromatic includes heterocycle. “Heterocycle” or“heteroaryl” means an aromatic group wherein 1 to 5 of the indicatedcarbon atoms are replaced by a heteroatom chosen from nitrogen, oxygen,sulfur, phosphorus, boron and silicon wherein the atom with the freevalence is a member of an aromatic ring, and any heterocyclic ketone andthioketone derivative thereof. Thus, heterocycle includes thienyl,furyl, pyrrolyl, pyrimidinyl, oxalyl, indolyl, purinyl, quinolyl,isoquinolyl, thiazolyl, imidozyl, etc.

Importantly, the Y aromatic groups of the conductive oligomer may bedifferent, i.e. the conductive oligomer may be a heterooligomer. Thatis, a conductive oligomer may comprise a oligomer of a single type of Ygroups, or of multiple types of Y groups. Thus, in a preferredembodiment, when a barrier monolayer is used as is described below, oneor more types of Y groups are used in the conductive oligomer within themonolayer with a second type(s) of Y group used above the monolayerlevel. Thus, as is described herein, the conductive oligomer maycomprise Y groups that have good packing efficiency within the monolayerat the electrode surface, and a second type(s) of Y groups with greaterflexibility and hydrophilicity above the monolayer level to facilitatenucleic acid hybridization. For example, unsubstituted benzyl rings maycomprise the Y rings for monolayer packing, and substituted benzyl ringsmay be used above the monolayer. Alternatively, heterocylic rings,either substituted or unsubstituted, may be used above the monolayer.Additionally, in one embodiment, heterooligomers are used even when theconductive oligomer does not extend out of the monolayer.

The aromatic group may be substituted with a substitution group,generally depicted herein as R. R groups may be added as necessary toaffect the packing of the conductive oligomers, i.e. when the nucleicacids attached to the conductive oligomers form a monolayer on theelectrode, R groups may be used to alter the association of theoligomers in the monolayer. R groups may also be added to 1) alter thesolubility of the oligomer or of compositions containing the oligomers;2) alter the conjugation or electrochemical potential of the system; and3) alter the charge or characteristics at the surface of the monolayer.

In a preferred embodiment, when the conductive oligomer is greater thanthree subunits, R groups are preferred to increase solubility whensolution synthesis is done. However, the R groups, and their positions,are chosen to minimally effect the packing of the conductive oligomerson a surface, particularly within a monolayer, as described below. Ingeneral, only small R groups are used within the monolayer, with largerR groups generally above the surface of the monolayer. Thus for examplethe attachment of methyl groups to the portion of the conductiveoligomer within the monolayer to increase solubility is preferred, withattachment of longer alkoxy groups, for example, C3 to C10, ispreferably done above the monolayer surface. In general, for the systemsdescribed herein, this generally means that attachment of stericallysignificant R groups is not done on any of the first three oligomersubunits, depending on the length of the insulator molecules.

Suitable R groups include, but are not limited to, hydrogen, alkyl,alcohol, aromatic, amino, amido, nitro, ethers, esters, aldehydes,sulfonyl, silicon moieties, halogens, sulfur containing moieties,phosphorus containing moieties, and ethylene glycols. In the structuresdepicted herein, R is hydrogen when the position is unsubstituted. Itshould be noted that some positions may allow two substitution groups, Rand R′, in which case the R and R′ groups may be either the same ordifferent.

By “alkyl group” or grammatical equivalents herein is meant a straightor branched chain alkyl group, with straight chain alkyl groups beingpreferred. If branched, it may be branched at one or more positions, andunless specified, at any position. The alkyl group may range from about1 to about 30 carbon atoms (C1-C30), with a preferred embodimentutilizing from about 1 to about 20 carbon atoms (C1-C20), with about C1through about C12 to about C15 being preferred, and C1 to C5 beingparticularly preferred, although in some embodiments the alkyl group maybe much larger. Also included within the definition of an alkyl groupare cycloalkyl groups such as C5 and C6 rings, and heterocyclic ringswith nitrogen, oxygen, sulfur or phosphorus. Alkyl also includesheteroalkyl, with heteroatoms of sulfur, oxygen, nitrogen, and siliconebeing preferred. Alkyl includes substituted alkyl groups. By“substituted alkyl group” herein is meant an alkyl group furthercomprising one or more substitution moieties “R”, as defined above.

By “amino groups” or grammatical equivalents herein is meant —NH₂, —NHRand —NR₂ groups, with R being as defined herein.

By “nitro group” herein is meant an —NO₂ group.

By “sulfur containing moieties” herein is meant compounds containingsulfur atoms, including but not limited to, thia-, thio- andsulfo-compounds, thiols (—SH and —SR), and sulfides (—RSR—). By“phosphorus containing moieties” herein is meant compounds containingphosphorus, including, but not limited to, phosphines and phosphates. By“silicon containing moieties” herein is meant compounds containingsilicon.

By “ether” herein is meant an —O—R group. Preferred ethers includealkoxy groups, with —O—(CH₂)₂CH₃ and —O—(CH₂)₄CH₃ being preferred.

By “ester” herein is meant a —COOR group.

By “halogen” herein is meant bromine, iodine, chlorine, or fluorine.Preferred substituted alkyls are partially or fully halogenated alkylssuch as CF₃, etc.

By “aldehyde” herein is meant —RCOH groups.

By “alcohol” herein is meant —OH groups, and alkyl alcohols —ROH.

By “amido” herein is meant —RCONH— or RCONR— groups.

By “ethylene glycol” herein is meant a —(O—CH₂—CH₂)_(n)— group, althougheach carbon atom of the ethylene group may also be singly or doublysubstituted, i.e. —(O—CR₂—CR₂)_(n)—, with R as described above. Ethyleneglycol derivatives with other heteroatoms in place of oxygen (i.e.—(N—CH₂—CH₂)_(n)— or —(S—CH₂—CH₂)_(n)—, or with substitution groups) arealso preferred.

Preferred substitution groups include, but are not limited to, methyl,ethyl, propyl, alkoxy groups such as —O—(CH₂)₂CH₃ and —O—(CH₂)₄CH₃ andethylene glycol and derivatives thereof.

Preferred aromatic groups include, but are not limited to, phenyl,naphthyl, naphthalene, anthracene, phenanthroline, pyrole, pyridine,thiophene, porphyrins, and substituted derivatives of each of these,included fused ring derivatives.

In the conductive oligomers depicted herein, when g is 1, B—D is a bondlinking two atoms or chemical moieties. In a preferred embodiment, B—Dis a conjugated bond, containing overlapping or conjugated π-orbitals.

Preferred B—D bonds are selected from acetylene (—C≡C—, also calledalkyne or ethyne), alkene (—CH═CH—, also called ethylene), substitutedalkene (—CR═CR—, —CH═CR— and —CR═CH—), amide (—NH—CO— and —NR—CO— or—CO—NH— and —CO—NR—), azo (—N═N—), esters and thioesters (—CO—O—,—O—CO—, —CS—O— and —O—CS—) and other conjugated bonds such as (—CH═N—,—CR═N—, —N═CH— and —N═CR—), (—SiH═SiH—, —SiR═SiH—, —SiR═SiH—, and—SiR═SiR—), (—SiH═CH—, —SiR═CH—, —SiH═CR—, —SiR═CR—, —CH═SiH—, —CR═SiH—,—CH═SiR—, and —CR═SiR—). Particularly preferred B—D bonds are acetylene,alkene, amide, and substituted derivatives of these three, and azo.Especially preferred B—D bonds are acetylene, alkene and amide. Theoligomer components attached to double bonds may be in the trans or cisconformation, or mixtures. Thus, either B or D may include carbon,nitrogen or silicon. The substitution groups are as defined as above forR.

When g=0 in the Structure 2 conductive oligomer, e is preferably 1 andthe D moiety may be carbonyl or a heteroatom moiety as defined above.

As above for the Y rings, within any single conductive oligomer, the B—Dbonds (or D moieties, when g=0) may be all the same, or at least one maybe different. For example, when m is zero, the terminal B—D bond may bean amide bond, and the rest of the B—D bonds may be acetylene bonds.Generally, when amide bonds are present, as few amide bonds as possibleare preferable, but in some embodiments all the B—D bonds are amidebonds. Thus, as outlined above for the Y rings, one type of B—D bond maybe present in the conductive oligomer within a monolayer as describedbelow, and another type above the monolayer level, to give greaterflexibility for nucleic acid hybridization.

In the structures depicted herein, n is an integer from 1 to 50,although longer oligomers may also be used (see for example Schumm etal., Angew. Chem. Int. Ed. Engl. 1994 33(13):1360). Without being boundby theory, it appears that for efficient hybridization of nucleic acidson a surface, the hybridization should occur at a distance from thesurface, i.e. the kinetics of hybridization increase as a function ofthe distance from the surface, particularly for long oligonucleotides of200 to 300 basepairs. Accordingly, the length of the conductive oligomeris such that the closest nucleotide of the nucleic acid is positionedfrom about 6 Å to about 100 Å (although distances of up to 500 Å may beused) from the electrode surface, with from about 25 Å to about 60 Åbeing preferred. Accordingly, n will depend on the size of the aromaticgroup, but generally will be from about 1 to about 20, with from about 2to about 15 being preferred and from about 3 to about 10 beingespecially preferred.

In the structures depicted herein, m is either 0 or 1. That is, when mis 0, the conductive oligomer may terminate in the B—D bond or D moiety,i.e. the D atom is attached to the nucleic acid either directly or via alinker. In some embodiments, for example when the conductive oligomer isattached to a phosphate of the ribose-phosphate backbone of a nucleicacid, there may be additional atoms, such as a linker, attached betweenthe conductive oligomer and the nucleic acid. Additionally, as outlinedbelow, the D atom may be the nitrogen atom of the amino-modified ribose.Alternatively, when m is 1, the conductive oligomer may terminate in Y,an aromatic group, i.e. the aromatic group is attached to the nucleicacid or linker.

As will be appreciated by those in the art, a large number of possibleconductive oligomers may be utilized. These include conductive oligomersfalling within the Structure 2 and Structure 9 formulas, as well asother conductive oligomers, as are generally known in the art, includingfor example, compounds comprising fused aromatic rings or Teflon®-likeoligomers, such as —(CF₂)_(n)—, —(CHF)_(n)— and —(CFR)_(n)—. See forexample, Schumm et al., angew. Chem. Intl. Ed. Engl. 33:1361 (1994);Grosshenny et al., Platinum Metals Rev. 40(1):26-35 (1996); Tour, Chem.Rev. 96:537-553 (1996); Hsung et al., Organometallics 14:4808-4815(1995; and references cited therein, all of which are expresslyincorporated by reference.

Particularly preferred conductive oligomers of this embodiment aredepicted below:

Structure 3 is Structure 2 when g is 1. Preferred embodiments ofStructure 3 include: e is zero, Y is pyrole or substituted pyrole; e iszero, Y is thiophene or substituted thiophene; e is zero, Y is furan orsubstituted furan; e is zero, Y is phenyl or substituted phenyl; e iszero, Y is pyridine or substituted pyridine; e is 1, B—D is acetyleneand Y is phenyl or substituted phenyl (see Structure 5 below). Apreferred embodiment of Structure 3 is also when e is one, depicted asStructure 4 below:

Preferred embodiments of Structure 4 are: Y is phenyl or substitutedphenyl and B—D is azo; Y is phenyl or substituted phenyl and B—D isalkene; Y is pyridine or substituted pyridine and B—D is acetylene; Y isthiophene or substituted thiophene and B—D is acetylene; Y is furan orsubstituted furan and B—D is acetylene; Y is thiophene or furan (orsubstituted thiophene or furan) and B—D are alternating alkene andacetylene bonds.

Most of the structures depicted herein utilize a Structure 4 conductiveoligomer. However, any Structure 4 oligomers may be substituted with aStructure 2, 3 or 9 oligomer, or other conducting oligomer, and the useof such Structure 4 depiction is not meant to limit the scope of theinvention.

Particularly preferred embodiments of Structure 4 include Structures 5,6, 7 and 8, depicted below:

Particularly preferred embodiments of Structure 5 include: n is two, mis one, and R is hydrogen; n is three, m is zero, and R is hydrogen; andthe use of R groups to increase solubility.

When the B—D bond is an amide bond, as in Structure 6, the conductiveoligomers are pseudopeptide oligomers. Although the amide bond inStructure 6 is depicted with the carbonyl to the left, i.e. —CONH—, thereverse may also be used, i.e. —NHCO—. Particularly preferredembodiments of Structure 6 include: n is two, m is one, and R ishydrogen; n is three, m is zero, and R is hydrogen (in this embodiment,the terminal nitrogen (the D atom) may be the nitrogen of theamino-modified ribose); and the use of R groups to increase solubility.

Preferred embodiments of Structure 7 include the first n is two, secondn is one, m is zero, and all R groups are hydrogen, or the use of Rgroups to increase solubility.

Preferred embodiments of Structure 8 include: the first n is three, thesecond n is from 1-3, with m being either 0 or 1, and the use of Rgroups to increase solubility.

In a preferred embodiment, the conductive oligomer has the structuredepicted in Structure 9:

In this embodiment, C are carbon atoms, n is an integer from 1 to 50, mis 0 or 1, J is a heteroatom selected from the group consisting ofnitrogen, silicon, phosphorus, sulfur, carbonyl or sulfoxide, and G is abond selected from alkane, alkene or acetylene, such that together withthe two carbon atoms the C-G-C group is an alkene (—CH═CH—), substitutedalkene (—CR═CR—) or mixtures thereof (—CH═CR— or —CR═CH—), acetylene(—C≡C—), or alkane (—CR₂—CR₂—, with R being either hydrogen or asubstitution group as described herein). The G bond of each subunit maybe the same or different than the G bonds of other subunits; that is,alternating oligomers of alkene and acetylene bonds could be used, etc.However, when G is an alkane bond, the number of alkane bonds in theoligomer should be kept to a minimum, with about six or less sigma bondsper conductive oligomer being preferred. Alkene bonds are preferred, andare generally depicted herein, although alkane and acetylene bonds maybe substituted in any structure or embodiment described herein as willbe appreciated by those in the art.

In a preferred embodiment, the m of Structure 9 is zero. In aparticularly preferred embodiment, m is zero and G is an alkene bond, asis depicted in Structure 10:

The alkene oligomer of structure 10, and others depicted herein, aregenerally depicted in the preferred trans configuration, althougholigomers of cis or mixtures of trans and cis may also be used. Asabove, R groups may be added to alter the packing of the compositions onan electrode, the hydrophilicity or hydrophobicity of the oligomer, andthe flexibility, i.e. the rotational, torsional or longitudinalflexibility of the oligomer. n is as defined above.

In a preferred embodiment, R is hydrogen, although R may be also alkylgroups and polyethylene glycols or derivatives.

In an alternative embodiment, the conductive oligomer may be a mixtureof different types of oligomers, for example of structures 2 and 9.

The conductive oligomers are covalently attached to the nucleic acids.By “covalently attached” herein is meant that two moieties are attachedby at least one bond, including sigma bonds, pi bonds and coordinationbonds.

The nucleic acid is covalently attached to the conductive oligomer, andthe conductive oligomer is also covalently attached to the electrode. Ingeneral, the covalent attachments are done in such a manner as tominimize the amount of unconjugated sigma bonds an electron must travelfrom the electron donor to the electron acceptor. Thus, linkers aregenerally short, or contain conjugated bonds with few sigma bonds.

The covalent attachment of the nucleic acid and the conductive oligomermay be accomplished in several ways. In a preferred embodiment, theattachment is via attachment to the base of the nucleoside, viaattachment to the backbone of the nucleic acid (either the ribose, thephosphate, or to an analogous group of a nucleic acid analog backbone),or via a transition metal ligand, as described below. The techniquesoutlined below are generally described for naturally occuring nucleicacids, although as will be appreciated by those in the art, similartechniques may be used with nucleic acid analogs.

In a preferred embodiment, the conductive oligomer is attached to thebase of a nucleoside of the nucleic acid. This may be done in severalways, depending on the oligomer, as is described below. In oneembodiment, the oligomer is attached to a terminal nucleoside, i.e.either the 3′ or 5′ nucleoside of the nucleic acid. Alternatively, theconductive oligomer is attached to an internal nucleoside.

The point of attachment to the base will vary with the base. Whileattachment at any position is possible, it is preferred to attach atpositions not involved in hydrogen bonding to the complementary base.Thus, for example, generally attachment is to the 5 or 6 position ofpyrimidines such as uridine, cytosine and thymine. For purines such asadenine and guanine, the linkage is preferably via the 8 position.Attachment to non-standard bases is preferably done at the comparablepositions.

In one embodiment, the attachment is direct; that is, there are nointervening atoms between the conductive oligomer and the base. In thisembodiment, for example, conductive oligomers with terminal acetylenebonds are attached directly to the base. Structure 11 is an example ofthis linkage, using a Structure 4 conductive oligomer and uridine as thebase, although other bases and conductive oligomers can be used as willbe appreciated by those in the art:

It should be noted that the pentose structures depicted herein may havehydrogen, hydroxy, phosphates or other groups such as amino groupsattached. In addition, the pentose and nucleoside structures depictedherein are depicted non-conventionally, as mirror images of the normalrendering. In addition, the pentose and nucleoside structures may alsocontain additional groups, such as protecting groups, at any position,for example as needed during synthesis.

In addition, the base may contain additional modifications as needed,i.e. the carbonyl or amine groups may be altered or protected, forexample as is depicted in FIG. 3 or 18.

In an alternative embodiment, the attachment is through an amide bondusing a linker as needed, as is generally depicted in Structure 12 usinguridine as the base and a Structure 4 oligomer:

Preferred embodiments of Structure 12 include Z is a methylene orethylene. The amide attachment can also be done using an amino group ofthe base, either a naturally occurring amino group such as in cytidineor adenidine, or from an amino-modified base as are known in the art.

In this embodiment, Z is a linker. Preferably, Z is a short linker ofabout 1 to about 5 atoms, that may or may not contain alkene bonds.Linkers are known in the art; for example, homo-or hetero-bifunctionallinkers as are well known (see 1994 Pierce Chemical Company catalog,technical section on cross-linkers, pages 155-200, incorporated hereinby reference). Preferred Z linkers include, but are not limited to,alkyl groups and alkyl groups containing heteroatom moieties, with shortalkyl groups, esters, epoxy groups and ethylene glycol and derivativesbeing preferred, with propyl, acetylene, and C₂ alkene being especiallypreferred. Z may also be a sulfone group, forming sulfonamide linkagesas discussed below.

In a preferred embodiment, the attachment of the nucleic acid and theconductive oligomer is done via attachment to the backbone of thenucleic acid. This may be done in a number of ways, including attachmentto a ribose of the ribose-phosphate backbone, or to the phosphate of thebackbone, or other groups of analogous backbones.

As a preliminary matter, it should be understood that the site ofattachment in this embodiment may be to a 3′ or 5′ terminal nucleotide,or to an internal nucleotide, as is more fully described below.

In a preferred embodiment, the conductive oligomer is attached to theribose of the ribose-phosphate backbone. This may be done in severalways. As is known in the art, nucleosides that are modified at eitherthe 2′ or 3′ position of the ribose with amino groups, sulfur groups,silicone groups, phosphorus groups, or oxo groups can be made (Imazawaet al., J. Org. Chem., 44:2039 (1979); Hobbs et al., J. Org. Chem.42(4):714 (1977); Verheyden et al., J. Orrg. Chem. 36(2):250 (1971);McGee et al., J. Org. Chem. 61:781-785 (1996); Mikhailopulo et al.,Liebigs. Ann. Chem. 513-519 (1993); McGee et al., Nucleosides &Nucleotides 14(6):1329 (1995), all of which are incorporated byreference). These modified nucleosides are then used to add theconductive oligomers.

A preferred embodiment utilizes amino-modified nucleosides. Theseamino-modified riboses can then be used to form either amide or aminelinkages to the conductive oligomers. In a preferred embodiment, theamino group is attached directly to the ribose, although as will beappreciated by those in the art, short linkers such as those describedherein for “Z” may be present between the amino group and the ribose.

In a preferred embodiment, an amide linkage is used for attachment tothe ribose. Preferably, if the conductive oligomer of Structures 2-4 isused, m is zero and thus the conductive oligomer terminates in the amidebond. In this embodiment, the nitrogen of the amino group of theamino-modified ribose is the “D” atom of the conductive oligomer. Thus,a preferred attachment of this embodiment is depicted in Structure 13(using the Structure 4 conductive oligomer):

As will be appreciated by those in the art, Structure 13 has theterminal bond fixed as an amide bond.

In a preferred embodiment, a heteroatom linkage is used, i.e. oxo,amine, sulfur, etc. A preferred embodiment utilizes an amine linkage.Again, as outlined above for the amide linkages, for amine linkages, thenitrogen of the amino-modified ribose may be the “D” atom of theconductive oligomer when the Structure 4 conductive oligomer is used.Thus, for example, Structures 14 and 15 depict nucleosides with theStructures 4 and 10 conductive oligomers, respectively, using thenitrogen as the heteroatom, although other heteroatoms can be used:

In Structure 14, preferably both m and t are not zero. A preferred Zhere is a methylene group, or other aliphatic alkyl linkers. One, two orthree carbons in this position are particularly useful for syntheticreasons; see FIG. 16.

In Structure 15, Z is as defined above. Suitable linkers includemethylene and ethylene.

In an alternative embodiment, the conductive oligomer is covalentlyattached to the nucleic acid via the phosphate of the ribose-phosphatebackbone (or analog) of a nucleic acid. In this embodiment, theattachment is direct, utilizes a linker or via an amide bond. Structure16 depicts a direct linkage, and Structure 17 depicts linkage via anamide bond (both utilize the Structure 4 conductive oligomer, althoughStructure 9 conductive oligomers are also possible). Structures 16 and17 depict the conductive oligomer in the 3′ position, although the 5′position is also possible. Furthermore, both Structures 16 and 17 depictnaturally occurring phosphodiester bonds, although as those in the artwill appreciate, non-standard analogs of phosphodiester bonds may alsobe used.

In Structure 16, if the terminal Y is present (i.e. m=1), thenpreferably Z is not present (i.e. t=0). If the terminal Y is notpresent, then Z is preferably present.

Structure 17 depicts a preferred embodiment, wherein the terminal B—Dbond is an amide bond, the terminal Y is not present, and Z is a linker,as defined herein.

In a preferred embodiment, the conductive oligomer is covalentlyattached to the nucleic acid via a transition metal ligand. In thisembodiment, the conductive oligomer is covalently attached to a ligandwhich provides one or more of the coordination atoms for a transitionmetal. In one embodiment, the ligand to which the conductive oligomer isattached also has the nucleic acid attached, as is generally depictedbelow in Structure 18. Alternatively, the conductive oligomer isattached to one ligand, and the nucleic acid is attached to anotherligand, as is generally depicted below in Structure 19. Thus, in thepresence of the transition metal, the conductive oligomer is covalentlyattached to the nucleic acid. Both of these structures depict Structure4 conductive oligomers, although other oligomers may be utilized.Structures 18 and 19 depict two representative structures:

In the structures depicted herein, M is a metal atom, with transitionmetals being preferred. Suitable transition metals for use in theinvention include, but are not limited to, cadmium (Cd), copper (Cu),cobalt (Co), palladium (Pd), zinc (Zn), iron (Fe), ruthenium (Ru),rhodium (Rh), osmium (Os), rhenium (Re), platinum (Pt), scandium (Sc),titanium (Ti), Vanadium (V), chromium (Cr), manganese (Mn), nickel (Ni),Molybdenum (Mo), technetium (Tc), tungsten (W), and iridium (Ir). Thatis, the first series of transition metals, the platinum metals (Ru, Rh,Pd, Os, Ir and Pt), along with Fe, Re, W, Mo and Tc, are preferred.Particularly preferred are ruthenium, rhenium, osmium, platinum, cobaltand iron.

L are the co-ligands, that provide the coordination atoms for thebinding of the metal ion. As will be appreciated by those in the art,the number and nature of the co-ligands will depend on the coordinationnumber of the metal ion. Mono-, di- or polydentate co-ligands may beused at any position. Thus, for example, when the metal has acoordination number of six, the L from the terminus of the conductiveoligomer, the L contributed from the nucleic acid, and r, add up to six.Thus, when the metal has a coordination number of six, r may range fromzero (when all coordination atoms are provided by the other two ligands)to four, when all the co-ligands are monodentate. Thus generally, r willbe from 0 to 8, depending on the coordination number of the metal ionand the choice of the other ligands.

In one embodiment, the metal ion has a coordination number of six andboth the ligand attached to the conductive oligomer and the ligandattached to the nucleic acid are at least bidentate; that is, r ispreferably zero, one (i.e. the remaining co-ligand is bidentate) or two(two monodentate co-ligands are used).

As will be appreciated in the art, the co-ligands can be the same ordifferent. Suitable ligands fall into two categories: ligands which usenitrogen, oxygen, sulfur, carbon or phosphorus atoms (depending on themetal ion) as the coordination atoms (generally referred to in theliterature as sigma (σ) donors) and organometallic ligands such asmetallocene ligands (generally referred to in the literature as pi (π)donors, and depicted herein as L_(m)) Suitable nitrogen donating ligandsare well known in the art and include, but are not limited to, NH₂; NHR;NRR′; pyridine; pyrazine; isonicotinamide; imidazole; bipyridine andsubstituted derivatives of bipyridine; terpyridine and substitutedderivatives; phenanthrolines, particularly 1,10-phenanthroline(abbreviated phen) and substituted derivatives of phenanthrolines suchas 4,7-dimethylphenanthroline and dipyridol[3,2-a:2′,3′-c]phenazine(abbreviated dppz); dipyridophenazine; 1,4,5,8,9,12-hexaazatriphenylene(abbreviated hat); 9,10-phenanethrenequinone diimine (abbreviated phi);1,4,5,8-tetraazaphenanthrene (abbreviated tap);1,4,8,11-tetra-azacyclotetradecane (abbreviated cyclam) and isocyanide.Substituted derivatives, including fused derivatives, may also be used.In some embodiments, porphyrins and substituted derivatives of theporphyrin family may be used. See for example, ComprehensiveCoordination Chemistry, Ed. Wilkinson et al., Pergammon Press, 1987,Chapters 13.2 (pp 73-98), 21.1 (pp. 813-898) and 21.3 (pp 915-957), allof which are hereby expressly incorporated by reference.

Suitable sigma donating ligands using carbon, oxygen, sulfur andphosphorus are known in the art. For example, suitable sigma carbondonors are found in Cotton and Wilkenson, Advanced Organic Chemistry,5th Edition, John Wiley & Sons, 1988, hereby incorporated by reference;see page 38, for example. Similarly, suitable oxygen ligands includecrown ethers, water and others known in the art. Phosphines andsubstituted phosphines are also suitable; see page 38 of Cotton andWilkenson.

The oxygen, sulfur, phosphorus and nitrogen-donating ligands areattached in such a manner as to allow the heteroatoms to serve ascoordination atoms.

In a preferred embodiment, organometallic ligands are used. In additionto purely organic compounds for use as redox moieties, and varioustransition metal coordination complexes with δ-bonded organic ligandwith donor atoms as heterocyclic or exocyclic substituents, there isavailable a wide variety of transition metal organometallic compoundswith π-bonded organic ligands (see Advanced Inorganic Chemistry, 5thEd., Cotton & Wilkinson, John Wiley & Sons, 1988, chapter 26;Organometallics, A Concise Introduction, Elschenbroich et al., 2nd Ed.,1992, VCH; and Comprehensive Organometallic Chemistry II, A Review ofthe Literature 1982-1994, Abel et al. Ed., Vol. 7, chapters 7, 8, 10 &11, Pergamon Press, hereby expressly incorporated by reference). Suchorganometallic ligands include cyclic aromatic compounds such as thecyclopentadienide ion [C₅H₅(−1)] and various ring substituted and ringfused derivatives, such as the indenylide (−1) ion, that yield a classof bis(cyclopentadieyl)metal compounds, (i.e. the metallocenes); see forexample Robins et al., J. Am. Chem. Soc. 104:1882-1893 (1982); andGassman et al., J. Am. Chem. Soc. 108:4228-4229 (1986), incorporated byreference. Of these, ferrocene [(C₅H₅)₂Fe] and its derivatives areprototypical examples which have been used in a wide variety of chemical(Connelly et al., Chem. Rev. 96:877-910 (1996), incorporated byreference) and electrochemical (Geiger et al., Advances inOrganometallic Chemistry 23:1-93; and Geiger et al., Advances inOrganometallic Chemistry 24:87, incorporated by reference) electrontransfer or “redox” reactions. Metallocene derivatives of a variety ofthe first, second and third row transition metals are potentialcandidates as redox moieties that are covalently attached to either theribose ring or the nucleoside base of nucleic acid. Other potentiallysuitable organometallic ligands include cyclic arenes such as benzene,to yield bis(arene)metal compounds and their ring substituted and ringfused derivatives, of which bis(benzene)chromium is a prototypicalexample, Other acyclic π-bonded ligands such as the allyl(−1) ion, orbutadiene yield potentially suitable organometallic compounds, and allsuch ligands, in conjuction with other π-bonded and δ-bonded ligandsconstitute the general class of organometallic compounds in which thereis a metal to carbon bond. Electrochemical studies of various dimers andoligomers of such compounds with bridging organic ligands, andadditional non-bridging ligands, as well as with and without metal-metalbonds are potential candidate redox moieties in nucleic acid analysis.

When one or more of the co-ligands is an organometallic ligand, theligand is generally attached via one of the carbon atoms of theorganometallic ligand, although attachment may be via other atoms forheterocyclic ligands. Preferred organometallic ligands includemetallocene ligands, including substituted derivatives and themetalloceneophanes (see page 1174 of Cotton and Wilkenson, supra). Forexample, derivatives of metallocene ligands such asmethylcyclopentadienyl, with multiple methyl groups being preferred,such as pentamethylcyclopentadienyl, can be used to increase thestability of the metallocene. In a preferred embodiment, only one of thetwo metallocene ligands of a metallocene are derivatized.

As described herein, any combination of ligands may be used. Preferredcombinations include: a) all ligands are nitrogen donating ligands; b)all ligands are organometallic ligands; and c) the ligand at theterminus of the conductive oligomer is a metallocene ligand and theligand provided by the nucleic acid is a nitrogen donating ligand, withthe other ligands, if needed, are either nitrogen donating ligands ormetallocene ligands, or a mixture. These combinations are depicted inrepresentative structures using the conductive oligomer of Structure 4are depicted in Structures 20 (using phenanthroline and amino asrepresentative ligands), 21 (using ferrocene as the metal-ligandcombination) and 22 (using cyclopentadienyl and amino as representativeligands).

In a preferred embodiment, the ligands used in the invention showaltered fluorescent properties depending on the redox state of thechelated metal ion. As described below, this thus serves as anadditional mode of detection of electron transfer through nucleic acid.

In a preferred embodiment, as is described more fully below, the ligandattached to the nucleic acid is an amino group attached to the 2′ or 3′position of a ribose of the ribose-phosphate backbone. This ligand maycontain a multiplicity of amino groups so as to form a polydentateligand which binds the metal ion. Other preferred ligands includecyclopentadiene and phenanthroline.

As described herein, the compositions described herein of nucleosidescovalently attached to conductive oligomers may be incorporated into alonger nucleic acid at any number of positions, including either the 5′or 3′ terminus of the nucleic acid or any internal position. As isoutlined below, this is generally done by adding a nucleotide with acovalently attached conductive oligomer to an oligonucleotide syntheticreaction at any position. After synthesis is complete, the nucleic acidwith the covalently attached conductive oligomer is attached to anelectrode. Thus, any number of additional nucleotides, modified or not,may be included at any position. Alternatively, the compositions aremade via post-nucleic acid synthesis modifications.

The total length of the nucleic acid will depend on its use. Generally,the nucleic acid compositions of the invention are useful asoligonucleotide probes. As is appreciated by those in the art, thelength of the probe will vary with the length of the target sequence andthe hybridization and wash conditions. Generally, oligonucleotide probesrange from about 8 to about 50 nucleotides, with from about 10 to about30 being preferred and from about 12 to about 25 being especiallypreferred. In some cases, very long probes may be used, e.g. 50 to200-300 nucleotides in length.

Also of consideration is the distance between the nucleoside containingthe electrode, i.e. a first electron transfer moiety, and the nucleosidecontaining a second electron transfer moiety. Electron transfer proceedsbetween the two electron transfer moieties. Since the rate of electrontransfer is distance dependent, the distance between the two electrontransfer moieties preferably ranges from about 1 to about 30 basepairs,with from about 2 to about 20 basepairs being preferred and from about 2to about 10 basepairs being particularly preferred and from about 2 to 6being especially preferred. However, probe specificity can be increasedby adding oligonucleotides on either side of the electron transfermoieties, thus increasing probe specificity without increasing thedistance an electron must travel.

Thus, in the structures depicted herein, nucleosides may be replacedwith nucleic acids.

In a preferred embodiment, the conductive oligomers with covalentlyattached nucleosides or nucleic acids as depicted herein are covalentlyattached to an electrode. Thus, one end or terminus of the conductiveoligomer is attached to the nucleoside or nucleic acid, and the other isattached to an electrode. In some embodiments it may be desirable tohave the conductive oligomer attached at a position other than aterminus, or even to have a branched conductive oligomer that isattached to an electrode at one terminus and to two or more nucleosidesat other termini, although this is not preferred. Similarly, theconductive oligomer may be attached at two sites to the electrode.

By “electrode” herein is meant a composition, which, when connected toan electronic device, is able to sense a current or charge and convertit to a signal. Thus, an electrode is an electron transfer moiety asdescribed herein. Preferred electrodes are known in the art and include,but are not limited to, certain metals and their oxides, including gold;platinum; palladium; silicon; aluminum; metal oxide electrodes includingplatinum oxide, titanium oxide, tin oxide, indium tin oxide, palladiumoxide, silicon oxide, aluminum oxide, molybdenum oxide (Mo₂O₆), tungstenoxide (WO₃) and ruthenium oxides; and carbon (including glassy carbonelectrodes, graphite and carbon paste). Preferred electrodes includegold, silicon, carbon and metal oxide electrodes.

The electrodes described herein are depicted as a flat surface, which isonly one of the possible conformations of the electrode and is forschematic purposes only. The conformation of the electrode will varywith the detection method used. For example, flat planar electrodes maybe preferred for optical detection methods, or when arrays of nucleicacids are made, thus requiring addressable locations for both synthesisand detection. Alternatively, for single probe analysis, the electrodemay be in the form of a tube, with the conductive oligomers and nucleicacids bound to the inner surface. This allows a maximum of surface areacontaining the nucleic acids to be exposed to a small volume of sample.

The covalent attachment of the conductive oligomer containing thenucleoside may be accomplished in a variety of ways, depending on theelectrode and the conductive oligomer used. Generally, some type oflinker is used, as depicted below as “A” in Structure 23, where X is theconductive oligomer, and the hatched surface is the electrode:

In this embodiment, A is a linker or atom. The choice of “A” will dependin part on the characteristics of the electrode. Thus, for example, Amay be a sulfur moiety when a gold electrode is used. Alternatively,when metal oxide electrodes are used, A may be a silicon (silane) moietyattached to the oxygen of the oxide (see for example Chen et al.,Langmuir 10:3332-3337 (1994); Lenhard et al., J. Electroanal. Chem.78:195-201 (1977), both of which are expressly incorporated byreference). When carbon based electrodes are used, A may be an aminomoiety (preferably a primary amine; see for example Deinhammer et al.,Langmuir 10:1306-1313 (1994)). Thus, preferred A moieties include, butare not limited to, silane moieties, sulfur moieties (including alkylsulfur moieties), and amino moieties. In a preferred embodiment, epoxidetype linkages with redox polymers such as are known in the art are notused.

Although depicted herein as a single moiety, the conductive oligomer maybe attached to the electrode with more than one “A” moiety; the “A”moieties may be the same or different. Thus, for example, when theelectrode is a gold electrode, and “A” is a sulfur atom or moiety, suchas generally depicted below in Structure 27, multiple sulfur atoms maybe used to attach the conductive oligomer to the electrode, such as isgenerally depicted below in Structures 24, 25 and 26. As will beappreciated by those in the art, other such structures can be made. InStructures 24, 25 and 26, the A moiety is just a sulfur atom, butsubstituted sulfur moieties may also be used.

It should also be noted that similar to Structure 26, it may be possibleto have a conductive oligomer terminating in a single carbon atom withthree sulfur moities attached to the electrode.

In a preferred embodiment, the electrode is a gold electrode, andattachment is via a sulfur linkage as is well known in the art, i.e. theA moiety is a sulfur atom or moiety. Although the exact characteristicsof the gold-sulfur attachment are not known, this linkage is consideredcovalent for the purposes of this invention. A representative structureis depicted in Structure 27. Structure 27 depicts the “A” linker ascomprising just a sulfur atom, although additional atoms may be present(i.e. linkers from the sulfur to the conductive oligomer or substitutiongroups).

In a preferred embodiment, the electrode is a carbon electrode, i.e. aglassy carbon electrode, and attachment is via a nitrogen of an aminegroup. A representative structure is depicted in Structure 28. Again,additional atoms may be present, i.e. Z type linkers.

In Structure 29, the oxygen atom is from the oxide of the metal oxideelectrode. The Si atom may also contain other atoms, i.e. be a siliconmoiety containing substitution groups.

Thus, in a preferred embodiment, electrodes are made that compriseconductive oligomers attached to nucleic acids for the purposes ofhybridization assays, as is more fully described herein. As will beappreciated by those in the art, electrodes can be made that have asingle species of nucleic acid, i.e. a single nucleic acid sequence, ormultiple nucleic acid species.

In addition, as outlined herein, the use of a solid support such as anelectrode enables the use of these gene probes in an array form. The useof oligonucleotide arrays are well known in the art. In addition,techniques are known for “addressing” locations within an electrode andfor the surface modification of electrodes. Thus, in a preferredembodiment, arrays of different nucleic acids are laid down on theelectrode, each of which are covalently attached to the electrode via aconductive linker. In this embodiment, the number of different probespecies of oligonucleotides may vary widely, from one to thousands, withfrom about 4 to about 100,000 being preferred, and from about 10 toabout 10,000 being particularly preferred.

In a preferred embodiment, the electrode further comprises a passayationagent, preferably in the form of a monolayer on the electrode surface.As outlined above, the efficiency of oligonucleotide hybridization mayincrease when the oligonucleotide is at a distance from the electrode. Apassayation agent layer facilitates the maintenance of the nucleic acidaway from the electrode surface. In addition, a passayation agent servesto keep charge carriers away from the surface of the electrode. Thus,this layer helps to prevent electrical contact between the electrodesand the electron transfer moieties, or between the electrode and chargedspecies within the solvent. Such contact can result in a direct “shortcircuit” or an indirect short circuit via charged species which may bepresent in the sample. Accordingly, the monolayer of passayation agentsis preferably tightly packed in a uniform layer on the electrodesurface, such that a minimum of “holes” exist. Alternatively, thepassayation agent may not be in the form of a monolayer, but may bepresent to help the packing of the conductive oligomers or othercharacteristics.

The passayation agents thus serve as a physical barrier to block solventaccessibility to the electrode. As such, the passayation agentsthemselves may in fact be either (1) conducting or (2) nonconducting,i.e. insulating, molecules. Thus, in one embodiment, the passayationagents are conductive oligomers, as described herein, with or without aterminal group to block or decrease the transfer of charge to theelectrode. Other passayation agents which may be conductive includeoligomers of —(CF₂)_(n)—, —(CHF)_(n)— and —(CFR)_(n)—. In a preferredembodiment, the passayation agents are insulator moieties.

An “insulator” is a substantially nonconducting oligomer, preferablylinear. By “substantially nonconducting” herein is meant that the rateof electron transfer through the insulator is slower than the rate ofelectron transfer through the stacked π-orbitals of double strandednucleic acid. Stated differently, the electrical resistance of theinsulator is higher than the electrical resistance of the nucleic acid.In a preferred embodiment, the rate of electron transfer through theinsulator is slower than or comparable to the rate through singlestranded nucleic acid. Similarly, the rate of electron transfer throughthe insulator is preferrably slower than the rate through the conductiveoligomers described herein. It should be noted however, as outlined inthe Examples, that even oligomers generally considered to be insulators,such as —(CH₂)₁₆ molecules, still may transfer electrons, albeit at aslow rate.

In a preferred embodiment, the insulators have a conductivity, S, ofabout 10⁻⁷ Ω⁻¹cm⁻¹ or lower, with less than about 10⁻⁸ Ω⁻¹ cm⁻¹ beingpreferred. See generally Gardner et al., supra.

Generally, insulators are alkyl or heteroalkyl oligomers or moietieswith sigma bonds, although any particular insulator molecule may containaromatic groups or one or more conjugated bonds. By “heteroalkyl” hereinis meant an alkyl group that has at least one heteroatom, i.e. nitrogen,oxygen, sulfur, phosphorus, silicon or boron included in the chain.Alternatively, the insulator may be quite similar to a conductiveoligomer with the addition of one or more heteroatoms or bonds thatserve to inhibit or slow, preferably substantially, electron transfer.

The passayation agents, including insulators, may be substituted with Rgroups as defined herein to alter the packing of the moieties orconductive oligomers on an electrode, the hydrophilicity orhydrophobicity of the insulator, and the flexibility, i.e. therotational, torsional or longitudinal flexibility of the insulator. Forexample, branched alkyl groups may be used. In addition, the terminus ofthe passayation agent, including insulators, may contain an additionalgroup to influence the exposed surface of the monolayer. For example,there may be negatively charged groups on the terminus to form anegatively charged surface such that when the nucleic acid is DNA or RNAthe nucleic acid is repelled or prevented from lying down on thesurface, to facilitate hybridization. Preferred passayation agentterminal groups include —NH₂, —OH, —COOH, and —CH₃.

The length of the passayation agent will vary as needed. As outlinedabove, it appears that hybridization is more efficient at a distancefrom the surface. Thus, the length of the passayation agents is similarto the length of the conductive oligomers, as outlined above. Inaddition, the conductive oligomers may be basically the same length asthe passayation agents or longer than them, resulting in the nucleicacids being more accessible to the solvent for hybridization.

The monolayer may comprise a single type of passayation agent, includinginsulators, or different types.

Suitable insulators are known in the art, and include, but are notlimited to, —(CH₂)_(n)—, —(CRH)_(n)—, and —(CR₂)_(n)—, ethylene glycolor derivatives using other heteroatoms in place of oxygen, i.e. nitrogenor sulfur (sulfur derivatives are not preferred when the electrode isgold).

The passayation agents are generally attached to the electrode in thesame manner as the conductive oligomer, and may use the same “A” linkeras defined above.

In a preferred embodiment, the compositions of the present inventioncomprise a conductive oligomer, covalently attached to both anelectrode, which serves as a first electron transfer moiety, and anucleic acid. In this embodiment, the conductive oligomer preferably hasthe structure depicted in Structures 2, 3, 4, 9 or 10. In thisembodiment, the compositions find use in general array-typetechnologies, i.e. the electrode may serve just as a solid support, withdetection proceeding using techniques well known in the art, such asfluorescence or radioisotope labelling.

In this embodiment, it is possible to have each nucleic acid be thesame, as an “anchor sequence”, such that a second sequence can be addedwhich contains the probe sequence and a sequence complementary to theanchor sequence. In this way, standard arrays of different anchorsequences can be made, which then can be used to generate custom arraysusing novel probe sequences linked to complementary anchor regions.

Similarly, it is possible to have compositions comprising electrodeswith conductive oligomers attached to probe nucleic acids, withoutsecond electron transfer moieties, and soluble second probe sequenceswith second electron transfer moieties. Upon binding of the targetsequence, which contains a first target domain for the first probesequence and a second target domain for the second probe sequence, whichpreferably are adjacent, electron transfer may occur.

Alternatively, it may be the target sequence which contains the secondelectron transfer moiety. Similar to methods which rely on amplificationand labelling of target sequences, the target nucleic acid may belabelled with a second electron transfer moiety which then can be usedto effect electron transfer upon formation of the hybridization complex.

In a preferred embodiment, the compositions of the present inventioncomprise a conductive oligomer, covalently attached to both anelectrode, which serves as a first electron transfer moiety, and anucleic acid, which has at least a second covalently attached electrontransfer moiety. As noted herein, the conductive oligomer and the secondelectron transfer moiety may be attached at any position of the nucleicacid.

In one embodiment, a nucleic acid is modified with more than twoelectron transfer moieties. For example, to increase the signal obtainedfrom the probe, or alter the required detector sensitivity, a pluralityof electron transfer moieties may be used. See PCT publication WO95/15971. For example, the conductive oligomer may be attached to aninternal nucleoside, with second electron transfer moieties (ETM)attached both 5′ and 3′ to the nucleoside containing the conductiveoligomer, as is generally depicted in Structure 29A. In one embodiment,the two additional electron transfer moieties are the same, and areplaced the same distance away from the conductive oligomer, to result ina uniform signal. Alternatively, the additional electron transfermoieties may be different and/or placed at different distances from theconductive oligomer.

The terms “electron donor moiety”, “electron acceptor moiety”, and“electron transfer moieties” or grammatical equivalents herein refers tomolecules capable of electron transfer under certain conditions. It isto be understood that electron donor and acceptor capabilities arerelative; that is, a molecule which can lose an electron under certainexperimental conditions will be able to accept an electron underdifferent experimental conditions. It is to be understood that thenumber of possible electron donor moieties and electron acceptormoieties is very large, and that one skilled in the art of electrontransfer compounds will be able to utilize a number of compounds in thepresent invention. Preferred electron transfer moieties include, but arenot limited to, transition metal complexes, organic electron transfermoieties, and electrodes.

In a preferred embodiment, the electron transfer moieties are transitionmetal complexes. Transition metals are those whose atoms have a partialor completed shell of electrons. Suitable transition metals for use inthe invention are listed above.

The transition metals are complexed with a variety of ligands, L,defined above, to form suitable transition metal complexes, as is wellknown in the art.

In addition to transition metal complexes, other organic electron donorsand acceptors may be covalently attached to the nucleic acid for use inthe invention. These organic molecules include, but are not limited to,riboflavin, xanthene dyes, azine dyes, acridine orange,N,N′-dimethyl-2,7-diazapyrenium dichloride (DAP²⁺), methylviologen,ethidium bromide, quinones such asN,N′-dimethylanthra(2,1,9-def:6,5,10-d′e′f′)diisoquinoline dichloride(ADIQ²⁺); porphyrins ([meso-tetrakis(N-methyl-x-pyridinium)porphyrintetrachloride], varlamine blue B hydrochloride, Bindschedler's green;2,6-dichloroindophenol, 2,6-dibromophenolindophenol; Brilliant crestblue (3-amino-9-dimethyl-amino-10-methylphenoxyazine chloride),methylene blue; Nile blue A (aminoaphthodiethylaminophenoxazinesulfate), indigo-5,5′,7,7′-tetrasulfonic acid, indigo-5,5′,7-trisulfonicacid; phenosafranine, indigo-5-monosulfonic acid; safranine T;bis(dimethylglyoximato)-iron(II) chloride; induline scarlet, neutralred, anthracene, coronene, pyrene, 9-phenylanthracene, rubrene,binaphthyl, DPA, phenothiazene, fluoranthene, phenanthrene, chrysene,1,8-diphenyl-1,3,5,7-octatetracene, naphthalene, acenaphthalene,perylene, TMPD and analogs and substituted derivatives of thesecompounds.

In one embodiment, the electron donors and acceptors are redox proteinsas are known in the art. However, redox proteins in many embodiments arenot preferred.

The choice of the specific electron transfer moieties will be influencedby the type of electron transfer detection used, as is generallyoutlined below.

In a preferred embodiment, these electron transfer moieties arecovalently attached to the nucleic acid in a variety of positions. In apreferred embodiment, the attachment is via attachment to the base ofthe nucleoside, or via attachment to the backbone of the nucleic acid,including either to a ribose of the ribose-phosphate backbone or to aphosphate moiety. In the preferred embodiments, the compositions of theinvention are designed such that the electron transfer moieties are asclose to the “π-way” as possible without significantly disturbing thesecondary and tertiary structure of the double helical nucleic acid,particularly the Watson-Crick basepairing. Alternatively, the attachmentcan be via a conductive oligomer, which is used as outlined above with anucleoside and an electrode; that is, an electron transfer moiety may becovalently attached to a conductive oligomer at one end and to anucleoside at the other, thus forming a general structure depicted inStructure 30:

In Structure 30, ETM is an electron transfer moiety, X is a conductiveoligomer, and q is an integer from zero to about 25, with preferred qbeing from about 2 to about 10. Additionally, linker moieties, forexample as are generally described herein as “Z”, may also be presentbetween the nucleoside and the conductive oligomer, and/or between theconductive oligomer and the electron transfer moiety. The depictednucleosides may be either terminal or internal nucleosides, and areusually separated by a number of nucleosides.

In a preferred embodiment, the second electron transfer moiety isattached to the base of a nucleoside, as is generally outlined above forattachment of the conductive oligomer. This is preferably done to thebase of an internal nucleoside. Surprisingly and unexpectedly, thisattachment does not perturb the Watson-Crick basepairing of the base towhich the electron transfer moiety is attached, as long as the moiety isnot too large. In fact, it appears that attachment at this site actuallyresults in less perturbation than attachment at the ribose of theribose-phosphate backbone, as measured by nucleic acid melting curves.Thus, when attachment to an internal base is done, the size of thesecond electron transfer moiety should be such that the structure ofdouble stranded nucleic acid containing the base-attached electrontransfer moiety is not significantly disrupted, and will not disrupt theannealing of single stranded nucleic acids. Preferrably, then, ligandsand full second electron transfer moieties are generally smaller thanthe size of the major groove of double stranded nucleic acid.

Alternatively, the second electron transfer moiety can be attached tothe base of a terminal nucleoside. Thus, when the target sequence to bedetected is n nucleosides long, a probe can be made which has the secondelectron transfer moiety attached at the n base. Alternatively, theprobe may contain an extra terminal nucleoside at an end of the nucleicacid (n+1 or n+2), which are used to covalently attach the electrontransfer moieties but which do not participate in basepairhybridization. Additionally, it is preferred that upon probehybridization, the terminal nucleoside containing the electron transfermoiety covalently attached at the base be directly adjacent toWatson-Crick basepaired nucleosides; that is, the electron transfermoiety should be as close as possible to the stacked π-orbitals of thebases such that an electron travels through a minimum of σ bonds toreach the “π-way”, or alternatively can otherwise electronically contactthe π-way.

The covalent attachment to the base will depend in part on the secondelectron transfer moiety chosen, but in general is similar to theattachment of conductive oligomers to bases, as outlined above. In apreferred embodiment, the second electron transfer moiety is atransition metal complex, and thus attachment of a suitable metal ligandto the base leads to the covalent attachment of the electron transfermoiety. Alternatively, similar types of linkages may be used for theattachment of organic electron transfer moieties, as will be appreciatedby those in the art.

In one embodiment, the C4 attached amino group of cytosine, the C6attached amino group of adenine, or the C2 attached amino group ofguanine may be used as a transition metal ligand, although in thisembodiment attachment at a terminal base is preferred since attachmentat these positions will perturb Watson-Crick basepairing.

Ligands containing aromatic groups can be attached via acetylenelinkages as is known in the art (see Comprehensive Organic Synthesis,Trost et al., Ed., Pergamon Press, Chapter 2.4: Coupling ReactionsBetween sp² and sp Carbon Centers, Sonogashira, pp 521-549, and pp950-953, hereby incorporated by reference). Structure 31 depicts arepresentative structure in the presence of the metal ion and any othernecessary ligands; Structure 31 depicts uridine, although as for all thestructures herein, any other base may also be used.

L_(a) is a ligand, which may include nitrogen, oxygen, sulfur orphosphorus donating ligands or organometallic ligands such asmetallocene ligands. Suitable L_(a) ligands include, but not limited to,phenanthroline, imidazole, bpy and terpy. L_(r) and M are as definedabove. Again, it will be appreciated by those in the art, a conductiveoligomer may be included between the nucleoside and the electrontransfer moiety.

Similarly, as for the conductive oligomers, the linkage may be doneusing a linker, which may utilize an amide linkage (see generally Telseret al., J. Am. Chem. Soc. 111:7221-7226 (1989); Telser et al., J. Am.Chem. Soc. 111:7226-7232 (1989), both of which are expresslyincorporated by reference). These structures are generally depictedbelow in Structure 32, which again uses uridine as the base, although asabove, the other bases may also be used:

In this embodiment, L is a ligand as defined above, with L_(r) and M asdefined above as well. Preferably, L is amino, phen, byp and terpy.

In a preferred embodiment, the second electron transfer moiety attachedto a nucleoside is a metallocene; i.e. the L and L_(r) of Structure 32are both metallocene ligands, L_(m), as described above. Structure 33depicts a preferred embodiment wherein the metallocene is ferrocene, andthe base is uridine, although other bases may be used:

Preferred metallocenes include ferrocene, cobaltocene and osmiumocene.

Thus, in a preferred embodiment, the invention provides metallocenescovalently attached to nucleosides. In a preferred embodiment, themetallocene is attached to the base of a nucleoside. In a preferredembodiment, the metallocene is ferrocene or substituted ferrocene.

In a preferred embodiment, the second electron transfer moiety isattached to a ribose at any position of the ribose-phosphate backbone ofthe nucleic acid, i.e. either the 5′ or 3′ terminus or any internalnucleoside. As is known in the art, nucleosides that are modified ateither the 2′ or 3′ position of the ribose can be made, with nitrogen,oxygen, sulfur and phosphorus-containing modifications possible.Amino-modified ribose is preferred. See generally PCT publication WO95/15971, incorporated herein by reference. These modification groupsmay be used as a transition metal ligand, or as a chemically functionalmoiety for attachment of other transition metal ligands andorganometallic ligands, or organic electron donor moieties as will beappreciated by those in the art. In this embodiment, a linker such asdepicted herein for “Z” may be used as well, or a conductive oligomerbetween the ribose and the electron transfer moiety. Preferredembodiments utilize attachment at the 2′ or 3′ position of the ribose,with the 2′ position being preferred. Thus for example, the conductiveoligomers depicted in Structure 13, 14 and 15 may be replaced byelectron transfer moieties; alternatively, as is depicted in Structure30, the electron transfer moieties may be added to the free terminus ofthe conductive oligomer.

In a preferred embodiment, a metallocene serves as the second electrontransfer moiety, and is attached via an amide bond as depicted below inStructure 34. The examples outline the synthesis of a preferred compoundwhen the metallocene is ferrocene.

Amine linkages, or linkages via other heteroatoms, are also possible.

In a preferred embodiment, the second electron transfer moiety isattached to a phosphate at any position of the ribose-phosphate backboneof the nucleic acid. This may be done in a variety of ways. In oneembodiment, phosphodiester bond analogs such as phosphoramide orphosphoramidite linkages may be incorporated into a nucleic acid as atransition metal ligand (see PCT publication WO 95/15971, incorporatedby reference). Alternatively, the conductive oligomers depicted inStructures 16 and 17 may be replaced by electron transfer moieties;alternatively, the electron transfer moieties may be added to the freeterminus of the conductive oligomer.

Preferred electron transfer moieties for covalent attachment to a singlestranded nucleic acid include, but are not limited to, transition metalcomplexes, including metallocenes and substituted metallocenes such asmetalloceneophanes, and complexes of Ru, Os, Re and Pt. Particularlypreferred are ferrocene and its derivatives (particularlypentamethylferrocene and ferroceneophane) and complexes of transitionmetals including Ru, Os, Re and Pt containing one or more amine orpolyamine, imidazole, phenathroline, pyridine, bipyridine and orterpyridine and their derivatives. For Pt, additional preferred ligandsinclude the diimine dithiolate complexes such asquinoxaline-2,3-dithiolate complexes.

As described herein, the invention provides compositions containingelectrodes as a first electron transfer moiety linked via a conductiveoligomer to a nucleic acid which has at least a second electron transfermoiety covalently attached. Any combination of positions of electrontransfer moiety attachment can be made; i.e. an electrode at the 5′terminus, a second electron transfer moiety at an internal position;electrode at the 5′ terminus, second moiety at the 3′ end; second moietyat the 5′ terminus, electrode at an internal position; both electrodeand second moiety at internal positions; electrode at an internalposition, second moiety at the 3′ terminus, etc. A preferred embodimentutilizes both the electrode and the second electron transfer moietyattached to internal nucleosides.

The compositions of the invention may additionally contain one or morelabels at any position. By “label” herein is meant an element (e.g. anisotope) or chemical compound that is attached to enable the detectionof the compound. Preferred labels are radioactive isotopic labels, andcolored or fluorescent dyes. The labels may be incorporated into thecompound at any position. In addition, the compositions of the inventionmay also contain other moieties such as cross-linking agents tofacilitate cross-linking of the target-probe complex. See for example,Lukhtanov et al., Nucl. Acids. Res. 24(4):683 (1996) and Tabone et al.,Biochem. 33:375 (1994), both of which are expressly incorporated byreference.

The compositions of the invention are generally synthesized as outlinedbelow, generally utilizing techniques well known in the art.

The compositions may be made in several ways. A preferred method firstsynthesizes a conductive oligomer attached to the nucleoside, withaddition of additional nucleosides followed by attachment to theelectrode. A second electron transfer moiety, if present, may be addedprior to attachment to the electrode or after. Alternatively, the wholenucleic acid may be made and then the completed conductive oligomeradded, followed by attachment to the electrode. Alternatively, theconductive oligomer and monolayer (if present) are attached to theelectrode first, followed by attachment of the nucleic acid. The lattertwo methods may be preferred when conductive oligomers are used whichare not stable in the solvents and under the conditions used intraditional nucleic acid synthesis.

In a preferred embodiment, the compositions of the invention are made byfirst forming the conductive oligomer covalently attached to thenucleoside, followed by the addition of additional nucleosides to form anucleic acid, including, if present, a nucleoside containing a secondelectron transfer moiety, with the last step comprising the addition ofthe conductive oligomer to the electrode.

The attachment of the conductive oligomer to the nucleoside may be donein several ways. In a preferred embodiment, all or part of theconductive oligomer is synthesized first (generally with a functionalgroup on the end for attachment to the electrode), which is thenattached to the nucleoside. Additional nucleosides are then added asrequired, with the last step generally being attachment to theelectrode. Alternatively, oligomer units are added one at a time to thenucleoside, with addition of additional nucleosides and attachment tothe electrode.

A general outline of a preferred embodiment is depicted in FIG. 1, usinga phenyl-acetylene oligomer as generally depicted in Structure 5. Otherconductive oligomers will be made using similar techniques, such asheterooligomers, or as known in the art. Thus, for example, conductiveoligomers using alkene or acetylene bonds are made as is known in theart.

The conductive oligomer is then attached to a nucleoside that maycontain one (or more) of the oligomer units, attached as depictedherein.

In a preferred embodiment, attachment is to a ribose of theribose-phosphate backbone. Thus, FIG. 1 depicts attachment via an amidelinkage, and FIGS. 2 and 16 depict the synthesis of compounds with aminelinkages. In a preferred embodiment, there is at least a methylene groupor other short aliphatic alkyl groups (as a Z group) between thenitrogen attached to the ribose and the aromatic ring of the conductiveoligomer. A representative synthesis is shown in FIG. 16.

Alternatively, attachment is via a phosphate of the ribose-phosphatebackbone. Examples of two synthetic schemes are shown in FIG. 4(synthesis of Structure 16 type compounds) and FIG. 5 (synthesis ofStructure 16 type compounds). Although both Figures show attachment atthe 3′ position of the ribose, attachment can also be made via the 2′position. In FIG. 5, Z is an ethylene linker, although other linkers maybe used as well, as will be appreciated by those in the art.

In a preferred embodiment, attachment is via the base. A general schemeis depicted in FIG. 3, using uridine as the nucleoside and aphenylene-acetylene conductive oligomer. As will be appreciated in theart, amide linkages are also possible, such as depicted in Structure 12,using techniques well known in the art. In a preferred embodiment,protecting groups may be added to the base prior to addition of theconductive oligomers, as is generally outlined in FIGS. 18 and 19. Inaddition, the palladium cross-coupling reactions may be altered toprevent dimerization problems; i.e. two conductive oligomers dimerizing,rather than coupling to the base.

Alternatively, attachment to the base may be done by making thenucleoside with one unit of the oligomer, followed by the addition ofothers.

Once the modified nucleosides are prepared, protected and activated,prior to attachment to the electrode, they may be incorporated into agrowing oligonucleotide by standard synthetic techniques (Gait,Oligonucleotide Synthesis: A Practical Approach, IRL Press, Oxford, UK1984; Eckstein) in several ways. In one embodiment, one or more modifiednucleosides are converted to the triphosphate form and incorporated intoa growing oligonucleotide chain by using standard molecular biologytechniques such as with the use of the enzyme DNA polymerase I, T4 DNApolymerase, T7 DNA polymerase, Taq DNA polymerase, reversetranscriptase, and RNA polymerases. For the incorporation of a 3′modified nucleoside to a nucleic acid, terminaldeoxynucleotidyltransferase may be used. (Ratliff, Terminaldeoxynucleotidyltransferase. In The Enzymes, Vol 14A. P. D. Boyer ed. pp105-118. Academic Press, San Diego, Calif. 1981). Alternatively, andpreferably, the amino nucleoside is converted to the phosphoramidite orH-phosphonate form, which are then used in solid-phase or solutionsyntheses of oligonucleotides. In this way the modified nucleoside,either for attachment at the ribose (i.e. amino- or thiol-modifiednucleosides) or the base, is incorporated into the oligonucleotide ateither an internal position or the 5′ terminus. This is generally donein one of two ways. First, the 5′ position of the ribose is protectedwith 4′,4-dimethoxytrityl (DMT) followed by reaction with either2-cyanoethoxy-bis-diisopropylaminophosphine in the presence ofdiisopropylammonium tetrazolide, or by reaction withchlorodiisopropylamino 2′-cyanoethyoxyphosphine, to give thephosphoramidite as is known in the art; although other techniques may beused as will be appreciated by those in the art. See Gait, supra;Caruthers, Science 230:281 (1985), both of which are expresslyincorporated herein by reference.

For attachment of an electron transfer moiety to the 3′ terminus, apreferred method utilizes the attachment of the modified nucleoside tocontrolled pore glass (CPG) or other oligomeric supports. In thisembodiment, the modified nucleoside is protected at the 5′ end with DMT,and then reacted with succinic anhydride with activation. The resultingsuccinyl compound is attached to CPG or other oligomeric supports as isknown in the art. Further phosphoramidite nucleosides are added, eithermodified or not, to the 5′ end after deprotection. Thus, the presentinvention provides conductive oligomers covalently attached tonucleosides attached to solid oligomeric supports such as CPG, andphosphoramidite derivatives of the nucleosides of the invention.

The growing nucleic acid chain may also comprise at least one nucleosidewith covalently attached second electron transfer moiety. As describedherein, modified nucleosides with covalently attached second electrontransfer moieties may be made, and incorporated into the nucleic acid asoutlined above for the conductive oligomer-nucleosides. When atransition metal complex is used as the second electron transfer moiety,synthesis may occur in several ways. In a preferred embodiment, theligand(s) are added to a nucleoside, followed by the transition metalion, and then the nucleoside with the transition metal complex attachedis added to an oligonucleotide, i.e. by addition to the nucleic acidsynthesizer. Alternatively, the ligand(s) may be attached, followed byincorporation into a growing oligonucleotide chain, followed by theaddition of the metal ion.

In a preferred embodiment, electron transfer moieties are attached to aribose of the ribose-phosphate backbone. This is generally done as isoutlined in PCT publication SO 95/15971, using amino-modifiednucleosides, at either the 2′ or 3′ position of the ribose. The aminogroup may then be used either as a ligand, for example as a transitionmetal ligand for attachment of the metal ion, or as a chemicallyfunctional group that can be used for attachment of other ligands ororganic electron transfer moieties, for example via amide linkages, aswill be appreciated by those in the art. For example, the examplesdescribe the synthesis of a nucleoside with a metallocene linked via anamide bond to the ribose.

In a preferred embodiment, electron transfer moieties are attached to aphosphate of the ribose-phosphate backbone. As outlined herein, this maybe done using phosphodiester analogs such as phosphoramidite bonds, seegenerally PCT publication WO 95/15971, or can be done in a similarmanner to that depicted in FIGS. 4 and 5, where the conductive oligomeris replaced by a transition metal ligand or complex or an organicelectron transfer moiety.

Attachment to alternate backbones, for example peptide nucleic acids oralternate phosphate linkages will be done as will be appreciated bythose in the art.

In a preferred embodiment, electron transfer moieties are attached to abase of the nucleoside. This may be done in a variety of ways. In oneembodiment, amino groups of the base, either naturally occurring oradded as is described herein (see the figures, for example), are usedeither as ligands for transition metal complexes or as a chemicallyfunctional group that can be used to add other ligands, for example viaan amide linkage, or organic electron transfer moieties. This is done aswill be appreciated by those in the art. Alternatively, nucleosidescontaining halogen atoms attached to the heterocyclic ring arecommercially available. Acetylene linked ligands may be added using thehalogenated bases, as is generally known; see for example, Tzalis etal., Tetrahedron Lett. 36(34):6017-6020 (1995); Tzalis et al.,Tetrahedron Lett. 36(2):3489-3490 (1995); and Tzalis et al., Chem.Communications (in press) 1996, all of which are hereby expresslyincorporated by reference. See also the examples, which describes thesynthesis of a metallocene attached via an acetylene linkage to thebase.

In one embodiment, the nucleosides are made with transition metalligands, incorporated into a nucleic acid, and then the transition metalion and any remaining necessary ligands are added as is known in theart. In an alternative embodiment, the transition metal ion andadditional ligands are added prior to incorporation into the nucleicacid.

In some embodiments, as outlined herein, conductive oligomers are usedbetween the second electron transfer moieties and the nucleosides. Theseare made using the techniques described herein, with the addition of theterminal second electron transfer moiety.

Once the nucleic acids of the invention are made, with a covalentlyattached conductive oligomer and optionally a second electron transfermoiety, the conductive oligomer is attached to the electrode. The methodwill vary depending on the type of electrode used. As is describedherein, the conductive oligomers are generally made with a terminal “A”linker to facilitate attachment to the electrode. For the purposes ofthis application, a sulfur-gold attachment is considered a covalentattachment.

In a preferred embodiment, conductive oligomers are covalently attachedvia sulfur linkages to the electrode. However, surprisingly, traditionalprotecting groups for use of attaching molecules to gold electrodes aregenerally ideal for use in both synthesis of the compositions describedherein and inclusion in oligonucleotide synthetic reactions.Accordingly, the present invention provides novel methods for theattachment of conductive oligomers to gold electrodes, utilizing unusualprotecting groups, including ethylpyridine, and trimethylsilylethyl asis depicted in the Figures.

This may be done in several ways. In a preferred embodiment, the subunitof the conductive oligomer which contains the sulfur atom for attachmentto the electrode is protected with an ethyl-pyridine ortrimethylsilylethyl group. For the former, this is generally done bycontacting the subunit containing the sulfur atom (preferably in theform of a sulfhydryl) with a vinyl pyridine group or vinyltrimethylsilylethyl group under conditions whereby an ethylpyridinegroup or trimethylsilylethyl group is added to the sulfur atom.

This subunit also generally contains a functional moiety for attachmentof additional subunits, and thus additional subunits are attached toform the conductive oligomer. The conductive oligomer is then attachedto a nucleoside, and additional nucleosides attached. The protectinggroup is then removed and the sulfur-gold covalent attachment is made.Alternatively, all or part of the conductive oligomer is made, and theneither a subunit containing a protected sulfur atom is added, or asulfur atom is added and then protected. The conductive oligomer is thenattached to a nucleoside, and additional nucleosides attached.Alternatively, the conductive oligomer attached to a nucleic acid ismade, and then either a subunit containing a protected sulfur atom isadded, or a sulfur atom is added and then protected. Alternatively, theethyl pyridine protecting group may be used as above, but removed afterone or more steps and replaced with a standard protecting group like adisulfide. Thus, the ethyl pyridine or trimethylsilylethyl group mayserve as the protecting group for some of the synthetic reactions, andthen removed and replaced with a traditional protecting group.

By “subunit” of a conductive polymer herein is meant at least the moietyof the conductive oligomer to which the sulfur atom is attached,although additional atoms may be present, including either functionalgroups which allow the addition of additional components of theconductive oligomer, or additional components of the conductiveoligomer. Thus, for example, when Structure 2 oligomers are used, asubunit comprises at least the first Y group.

A preferred method comprises 1) adding an ethyl pyridine ortrimethylsilylethyl protecting group to a sulfur atom attached to afirst subunit of a conductive oligomer, generally done by adding a vinylpyridine or trimethylsilylethyl group to a sulfhydryl; 2) addingadditional subunits to form the conductive oligomer; 3) adding at leasta first nucleoside to the conductive oligomer; 4) adding additionalnucleosides to the first nucleoside to form a nucleic acid; 5) attachingthe conductive oligomer to the gold electrode. This may also be done inthe absence of nucleosides, as is described in the Examples.

The above method may also be used to attach passayation molecules to agold electrode.

In a preferred embodiment, a monolayer of passayation agents is added tothe electrode. Generally, the chemistry of addition is similar to or thesame as the addition of conductive oligomers to the electrode, i.e.using a sulfur atom for attachment to a gold electrode, etc.Compositions comprising monolayers in addition to the conductiveoligomers covalently attached to nucleic acids (with or without secondelectron transfer moieties) may be made in at least one of five ways:(1) addition of the monolayer, followed by subsequent addition of theconductive oligomer-nucleic acid complex; (2) addition of the conductiveoligomer-nucleic acid complex followed by addition of the monolayer; (3)simultaneous addition of the monolayer and conductive oligomer-nucleicacid complex; (4) formation of a monolayer (using any of 1, 2 or 3)which includes conductive oligomers which terminate in a functionalmoiety suitable for attachment of a completed nucleic acid; or (5)formation of a monolayer which includes conductive oligomers whichterminate in a functional moiety suitable for nucleic acid synthesis,i.e. the nucleic acid is synthesized on the surface of the monolayer asis known in the art. Such suitable functional moieties include, but arenot limited to, nucleosides, amino groups, carboxyl groups, protectedsulfur moieties, or hydroxyl groups for phosphoramidite additions. Theexamples describe the formation of a monolayer on a gold electrode usingthe preferred method (1).

As will be appreciated by those in the art, electrodes may be made thathave any combination of nucleic acids, conductive oligomers andpassayation agents. Thus, a variety of different conductive oligomers orpassayation agents may be used on a single electrode.

Once made, the compositions find use in a number of applications, asdescribed herein. In a preferred embodiment, the compositions of theinvention are used as probes in hybridization assays to detect targetsequences in a sample. The term “target sequence” or grammaticalequivalents herein means a nucleic acid sequence on a single strand ofnucleic acid. The target sequence may be a portion of a gene, aregulatory sequence, genomic DNA, cDNA, RNA including mRNA and rRNA, orothers. It may be any length, with the understanding that longersequences are more specific. As will be appreciated by those in the art,the complementary target sequence may take many forms. For example, itmay be contained within a larger nucleic acid sequence, i.e. all or partof a gene or mRNA, a restriction fragment of a plasmid or genomic DNA,among others. As is outlined more fully below, probes are made tohybridize to target sequences to determine the presence or absence ofthe target sequence in a sample. Generally speaking, this term will beunderstood by those skilled in the art.

If required, the target sequence is prepared using known techniques. Forexample, the sample may be treated to lyse the cells, using known lysisbuffers, electroporation, etc., with purification and/or amplificationoccuring as needed, as will be appreciated by those in the art.

The probes of the present invention are designed to be complementary tothe target sequence, such that hybridization of the target sequence andthe probes of the present invention occurs. As outlined below, thiscomplementarity need not be perfect; there may be any number of basepair mismatches which will interfere with hybridization between thetarget sequence and the single stranded nucleic acids of the presentinvention. However, if the number of mutations is so great that nohybridization can occur under even the least stringent of hybridizationconditions, the sequence is not a complementary target sequence.

A variety of hybridization conditions may be used in the presentinvention, including high, moderate and low stringency conditions; seefor example Maniatis et al., Molecular Cloning: A Laboratory Manual, 2dEdition, 1989, and Short Protocols in Molecular Biology, ed. Ausubel, etal, hereby incorporated by reference. The hybridization conditions mayalso vary when a non-ionic backbone, i.e. PNA is used, as is known inthe art. In addition, cross-linking agents may be added after targetbinding to cross-link, i.e. covalently attach, the two strands of thehybridization complex.

In a preferred embodiment, single stranded nucleic acids are made whichcontain a first electron transfer moiety, an electrode, and at least asecond electron transfer moiety. Hybridization to a target sequenceforms a double stranded hybridization complex. In a hybridizationcomplex, at least the sequence between the nucleosides containing theelectron transfer moieties is double stranded, i.e. contains stackedπ-orbitals, such that upon initiation, the complex is capable oftransferring at least one electron from one of the electron transfermoieties to the other. As will be appreciated by those in the art, anelectrode may serve as either an electron donor or acceptor, and thechoice of the second electron transfer species is made accordingly.

In an alternative embodiment, compositions comprising a) a first singlestranded nucleic acid covalently attached to an electrode via aconductive oligomer and b) a second single stranded nucleic acidcontaining a second electron transfer moiety, are made. In thisembodiment, the first single stranded nucleic acid is capable ofhybridizing to a first target domain, and the second single strandednucleic acid is capable of hybridizing to a second target domain. Theterms “first target domain” and “second target domain” or grammaticalequivalents herein means two portions of a target sequence within anucleic acid which is under examination. The first target domain may bedirectly adjacent to the second target domain, or the first and secondtarget domains may be separated by an intervening target domain.Preferably, there are no gaps between the domains; i.e. they arecontiguous. The terms “first” and “second” are not meant to confer anorientation of the sequences with respect to the 5′-3′ orientation ofthe target sequence. For example, assuming a 5′-3′ orientation of thecomplementary target sequence, the first target domain may be locatedeither 5′ to the second domain, or 3′ to the second domain.

In this embodiment, the first single stranded nucleic acid is hybridizedto the first target domain, and the second single stranded nucleic acidis hybridized to the second target domain to form a hybridizationcomplex. As outlined above, the hybridization complex is then capable oftransferring at least one electron between the electron transfermoieties upon initiation.

In one embodiment, compositions comprising a) a single stranded nucleicacid covalently attached to an electrode via a conductive oligomer, andb) a target nucleic acid are made. In this embodiment, oncehybridization of the target and the probe occurs, a hybridizationindicator is added. Hybridization indicators serve as an electrontransfer moiety that will preferentially associate with double strandednucleic acid is added, usually reversibly, similar to the method ofMillan et al., Anal. Chem. 65:2317-2323 (1993); Millan et al., Anal.Chem. 662943-2948 (1994), both of which are hereby expresslyincorporated by reference. Hybridization indicators includeintercalators and minor and/or major groove binding moieties. In apreferred embodiment, intercalators may be used; since intercalationgenerally only occurs in the presence of double stranded nucleic acid,only in the presence of target hybridization will electron transferoccur. Intercalating transition metal complex electron transfer moietiesare known in the art. Similarly, major or minor groove binding moieties,such as methylene blue, may also be used in this embodiment.

In addition, hybridization indicators may be used in any or all of theother systems of the invention; for example, they may be added tofacilitate, quench or amplify the signal generated by the system, inaddition to the covalently attached electron transfer moieties. Forexample, it has been shown by Millan, above, that some hybridizationindicators may preferentially bind to perfectly complementary doublestranded nucleic acids over nucleic acids containing mismatches. Thiscould serve to contribute additional information about the system.Similarly, electronic coupling could be increased due to hybridizationindicator binding. Alternatively, quenching of the electron transfersignal could be achieved using hybridization indicators, whereby theelectrons would flow between the second electron transfer moiety and thehybridization indicator, rather than the electrode.

A further embodiment utilizes compositions comprising a) a first singlestranded nucleic acid covalently attached to an electrode via aconductive oligomer; b) a second single stranded nucleic acid containinga second electron transfer moiety; and c) an intervening single strandednucleic acid, which may or may not be labelled or contain an electrontransfer moiety. As generally outlined in PCT WO 95/15971, the firstsingle stranded nucleic acid hybridizes to the first target domain, thesecond single stranded nucleic acid hybridizes to the second targetdomain, and the intervening nucleic acid hybridizes to the interveningtarget domain, with electron transfer upon initiation. The interveningnucleic acid may be any length, taking into consideration the parametersfor the distance between the electron transfer moieties, although it maybe a single nucleoside.

In addition, the first and second, or first, intervening and second,nucleic acids may be ligated together prior to the electron transferreaction, using standard molecular biology techniques such as the use ofa ligase.

In one embodiment, the compositions of the invention are used to detectmismatches in a complementary target sequence. A mismatch, whether it bea substitution, insertion or deletion of a nucleoside or nucleosides,results in incorrect base pairing in a hybridized double helix ofnucleic acid. Accordingly, if the path of an electron from an electrondonor moiety to an electron acceptor moiety spans the region where themismatch lies, the electron transfer will be reduced such that a changein the relative impedance will be seen. Therefore, in this embodiment,the electron donor moiety is attached to the nucleic acid at a 5′position from the mutation, and the electron acceptor moiety is attachedat a 3′ position, or vice versa.

Electron transfer is generally initiated electronically, with voltagebeing preferred. A potential is applied to a sample containing modifiednucleic acid probes. Precise control and variations in the appliedpotential can be via a potentiostat and either a three electrode system(one reference, one sample and one counter electrode) or a two electrodesystem (one sample and one counter electrode). This allows matching ofapplied potential to peak electron transfer potential of the systemwhich depends in part on the choice of electron acceptors attached tothe nucleic acid and in part on the conductive oligomer used. Asdescribed herein, ferrocene is a preferred electron transfer moiety.

Preferably, initiation and detection is chosen to maximize the relativedifference between the impedances of double stranded nucleic acid andsingle stranded nucleic acid systems.

The efficiency of electron transfer through nucleic acid is a functionof the impedance of the compound.

In a preferred embodiment, a co-reductant or co-oxidant (collectively,co-redoxant) is used, as an additional electron source or sink. Seegenerally Sato et al., Bull. Chem. Soc. Jpn 66:1032 (1993); Uosaki etal., Electrochimica Acta 36:1799 (1991); and Alleman et al., J. Phys.Chem 100:17050 (1996); all of which are incorporated by reference.

In a preferred embodiment, an input electron source in solution is usedin the initiation of electron transfer, preferably when initiation anddetection are being done using DC current, and when a passayation agentmonolayer is present on the electrode. This may be done in severalgeneral ways. In a preferred embodiment, an input electron source isused that has a lower or similar redox potential than the secondelectron transfer moiety (ETM) covalently attached to the probe nucleicacid. Thus, at voltages above the redox potential of the input electronsource, both the second ETM and the input electron source are oxidizedand can thus donate electrons; the ETM donates through the hybridizationcomplex, through the conductive oligomer, to the electrode, and theinput source donates to the ETM. For example, ferrocene, as a second ETMattached to the compositions of the invention as described in theexamples, has a redox potential of roughly 200 mV in aqueous solution(which changes slightly depending on what the ferrocene is bound to).Ferrocyanide, an electron source, has a redox potential of roughly 200mV as well (in aqueous solution). Accordingly, at or above voltages ofroughly 200 mV, ferrocene is converted to ferricenium, which thentransfers an electron to the nucleic acid. If this nucleic acid isdouble stranded, transfer proceeds rapidly through the double strandednucleic acid, through the conductive oligomer, to the electrode. Now theferricyanide can be oxidized to transfer an electron to the ETM. In thisway, the electron source (or co-reductant) serves to amplify the signalgenerated in the system, as the electron source molecules rapidly andrepeatedly donate electrons to the second ETM attached to the nucleicacid. The rate of electron donation or acceptance will be limited by therate of diffusion of the co-reductant, which in turn is affected by theconcentration and size, etc.

Alternatively, input electron sources that have lower redox potentialsthan the second ETM are used. At voltages less than the redox potentialof the ETM, but higher than the redox potential of the electron source,the input source such as ferrocyanide is unable to be oxided and thus isunable to donate an electron to the ETM; i.e. no electron transferoccurs. The use of electron source molecules, however, is only possiblewhen an insulating or passayation layer is present, since otherwise thesource molecule will transfer electrons directly to the electrode.Accordingly, in a preferred embodiment, an electron source is used insolution to amplify the signal generated in the presence of hybridizedtarget sequence.

In an alternate preferred embodiment, an input electron source is usedthat has a higher redox potential than the second electron transfermoiety (ETM) covalently attached to the probe nucleic acid. For example,luminol, an electron source, has a redox potential of roughly 720 mV. Atvoltages higher than the redox potential of the ETM, but lower than theredox potential of the electron source, i.e. 200-720 mV, the ferroceneis oxided, and transfers a single electron to the electrode via theconductive oligomer. However, the ETM is unable to accept any electronsfrom the luminol electron source, since the voltages are less than theredox potential of the luminol. However, at or above the redox potentialof luminol, the luminol then transfers an electron to the ETM, allowingrapid and repeated electron transfer. In this way, the electron source(or co-reductant) serves to amplify the signal generated in the system,as the electron source molecules rapidly and repeatedly donate electronsto the second ETM attached to the nucleic acid.

Luminol has the added benefit of becoming a chemiluminescent speciesupon oxidation (see Jirka et al., Analytica Chimica Acta 284:345(1993)), thus allowing photo-detection of electron transfer throughdouble-stranded nucleic acid. Thus, as long as the luminol is unable tocontact the electrode directly, i.e. in the presence of a passayationlayer, luminol can only be oxidized by transferring an electron to thesecond electron transfer moiety on the nucleic acid (e.g. ferrocene).When double stranded nucleic acid is not present, i.e. when the targetsequence is not hybridized to the composition of the invention, thesystem has a high impedance, resulting in a low photon emission and thusa low (if any) signal from the luminol. In the presence of doublestranded nucleic acid, i.e. target sequence hybridization, the secondelectron transfer moieties have low impedance, thus generating a muchlarger signal. Thus, the measure of luminol oxidation by photon emissionis an indirect measurement of the ability of the second electrontransfer moiety to donate electrons to the electrode. Furthermore, sincephoton detection is generally more sensitive than electronic detection,the sensitivity of the system may be increased. Initial results suggestthat luminescence may depend on hydrogen peroxide concentration, pH, andluminol concentration, the latter of which appears to be non-linear.

Suitable electron source molecules are well known in the art, andinclude, but are not limited to, ferricyanide, and luminol.

Alternatively, output electron acceptors or sinks could be used, i.e.the above reactions could be run in reverse, with the ETM such as ametallocene receiving an electron from the electrode, converting it tothe metallicenium, with the output electron acceptor then accepting theelectron rapidly and repeatedly. In this embodiment, cobalticenium isthe preferred ETM.

Electron transfer through nucleic acid can be detected in a variety ofways. A variety of detection methods may be used, including, but notlimited to, optical detection, which includes fluorescence,phosphorescence, luminiscence, chemiluminescence,electrochemiluminescence, and refractive index; and electronicdetection, including, but not limited to, amperommetry, voltammetry,capacitance and impedence. These methods include time or frequencydependent methods based on AC or DC currents, pulsed methods, lock-intechniques, filtering (high pass, low pass, band pass), andtime-resolved techniques including time-resolved fluorescence. In someembodiments, all that is required is electron transfer detection; inothers, the rate of electron transfer may be determined.

In one embodiment, the efficient transfer of electrons from one end of anucleic acid double helix to the other results in stereotyped changes inthe redox state of both the electron donor and acceptor. With manyelectron transfer moieties including the complexes of rutheniumcontaining bipyridine, pyridine and imidazole rings, these changes inredox state are associated with changes in spectral properties.Significant differences in absorbance are observed between reduced andoxidized states for these molecules. See for example Fabbrizzi et al.,Chem. Soc. Rev. 1995 pp 197-202). These differences can be monitoredusing a spectrophotometer or simple photomultiplier tube device.

In this embodiment, possible electron donors and acceptors include allthe derivatives listed above for photoactivation or initiation.Preferred electron donors and acceptors have characteristically largespectral changes upon oxidation and reduction resulting in highlysensitive monitoring of electron transfer. Such examples includeRu(NH₃)₄py and Ru(bpy)₂im as preferred examples. It should be understoodthat only the donor or acceptor that is being monitored by absorbanceneed have ideal spectral characteristics. That is, the electron acceptorcan be optically invisible if only the electron donor is monitored forabsorbance changes.

In a preferred embodiment, the electron transfer is detectedfluorometrically. Numerous transition metal complexes, including thoseof ruthenium, have distinct fluorescence properties. Therefore, thechange in redox state of the electron donors and electron acceptorsattached to the nucleic acid can be monitored very sensitively usingfluorescence, for example with Ru(4,7-biphenyl₂-phenanthroline)₃ ²⁺. Theproduction of this compound can be easily measured using standardfluorescence assay techniques. For example, laser induced fluorescencecan be recorded in a standard single cell fluorimeter, a flow through“on-line” fluorimeter (such as those attached to a chromatographysystem) or a multi-sample “plate-reader” similar to those marketed for96-well immuno assays.

Alternatively, fluorescence can be measured using fiber optic sensorswith nucleic acid probes in solution or attached to the fiber optic.Fluorescence is monitored using a photomultiplier tube or other lightdetection instrument attached to the fiber optic. The advantage of thissystem is the extremely small volumes of sample that can be assayed.

In addition, scanning fluorescence detectors such as the FluorImagersold by Molecular Dynamics are ideally suited to monitoring thefluorescence of modified nucleic acid molecules arrayed on solidsurfaces. The advantage of this system is the large number of electrontransfer probes that can be scanned at once using chips covered withthousands of distinct nucleic acid probes.

Many transition metal complexes display fluorescence with large Stokesshifts. Suitable examples include bis- and trisphenanthroline complexesand bis- and trisbipyridyl complexes of transition metals such asruthenium (see Juris, A., Balzani, V., et. al. Coord. Chem. Rev., V. 84,p. 85-277, 1988). Preferred examples display efficient fluorescence(reasonably high quantum yields) as well as low reorganization energies.These include Ru(4,7-biphenyl₂-phenanthroline)₃ ²⁺,Ru(4,4′-diphenyl-2,2′-bipyridine)₃ ²⁺ and platinum complexes (seeCummings et al., J. Am. Chem. Soc. 118:1949-1960 (1996), incorporated byreference).

Alternatively, a reduction in fluorescence associated with hybridizationcan be measured using these systems. An electron transfer “donor”molecule that fluoresces readily when on single stranded nucleic acid(with an “acceptor” on the other end) will undergo a reduction influorescent intensity when complementary nucleic acid binds the probeallowing efficient transfer of the excited state electron. This drop influorescence can be easily monitored as an indicator of the presence ofa target sequence using the same methods as those above.

In a further embodiment, electrochemiluminescence is used as the basisof the electron transfer detection. With some electron transfer moietiessuch as Ru²⁺(bpy)₃, direct luminescence accompanies excited state decay.Changes in this property are associated with nucleic acid hybridizationand can be monitored with a simple photomultiplier tube arrangement (seeBlackburn, G. F. Clin. Chem. 37: 1534-1539 (1991); and Juris et al.,supra.

In a preferred embodiment, electronic detection is used, includingamperommetry, voltammetry, capacitance, and impedence. Suitabletechniques include, but are not limited to, electrogravimetry;coulometry (including controlled potential coulometry and constantcurrent coulometry); voltametry (cyclic voltametry, pulse voltametry(normal pulse voltametry, square wave voltametry, differential pulsevoltametry, Osteryoung square wave voltametry, and coulostatic pulsetechniques); stripping analysis (aniodic stripping analysis, cathiodicstripping analysis, square wave stripping voltammetry); conductancemeasurements (electrolytic conductance, direct analysis); time-dependentelectrochemical analyses (chronoamperometry, chronopotentiometry, cyclicchronopotentiometry and amperometry, AC polography, chronogalvametry,and chronocoulometry); AC impedance measurement; capacitancemeasurement; AC voltametry; and photoelectrochemistry.

In a preferred embodiment, monitoring electron transfer through nucleicacid is via amperometric detection. This method of detection involvesapplying a potential (as compared to a separate reference electrode)between the nucleic acid-conjugated electrode and a reference (counter)electrode in the sample containing target genes of interest. Electrontransfer of differing efficiencies is induced in samples in the presenceor absence of target nucleic acid; that is, the presence or absence ofthe target nucleic acid alters the impedance of the nucleic acid (i.e.double stranded versus single stranded) system which can result indifferent currents.

The device for measuring electron transfer amperometrically involvessensitive current detection and includes a means of controlling thevoltage potential, usually a potentiostat. This voltage is optimizedwith reference to the potential of the electron donating complex on thenucleic acid. Possible electron donating complexes include thosepreviously mentioned with complexes of iron, osmium, platinum, cobalt,rhenium and ruthenium being preferred and complexes of iron being mostpreferred.

In a preferred embodiment, alternative electron detection modes areutilized. For example, potentiometric (or voltammetric) measurementsinvolve non-faradaic (no net current flow) processes and are utilizedtraditionally in pH and other ion detectors. Similar sensors are used tomonitor electron transfer through nucleic acid. In addition, otherproperties of insulators (such as resistance) and of conductors (such asconductivity, impedance and capacitance) could be used to monitorelectron transfer through nucleic acid. Finally, any system thatgenerates a current (such as electron transfer) also generates a smallmagnetic field, which may be monitored in some embodiments.

It should be understood that one benefit of the fast rates of electrontransfer observed in the compositions of the invention is that timeresolution can greatly enhance the signal-to-noise results of monitorsbased on absorbance, fluorescence and electronic current. The fast ratesof electron transfer of the present invention result both in highsignals and stereotyped delays between electron transfer initiation andcompletion. By amplifying signals of particular delays, such as throughthe use of pulsed initiation of electron transfer and “lock-in”amplifiers of detection, between two and four orders of magnitudeimprovements in signal-to-noise may be achieved.

In a preferred embodiment, electron transfer is initiated usingalternating current (AC) methods. Without being bound by theory, itappears that nucleic acids, bound to an electrode, generally respondsimilarly to an AC voltage resistor and capacitor in series. Basically,any methods which enable the determination of the nature of thesecomplexes, which act as a resistor and capacitor, can be used as thebasis of detection. Surprisingly, traditional electrochemical theory,such as exemplified in Laviron et al., J. Electroanal. Chem. 97:135(1979) and Laviron et al., J. Electroanal. Chem. 105:35 (1979), both ofwhich are incorporated by reference, do not accurately model the systemsdescribed herein, except for very small E_(AC) (less than 10 mV). Thatis, the AC current (I) is not accurately described by Laviron'sequation. This may be due in part to the fact that this theory assumesan unlimited source and sink of electrons, which is not true in thepresent systems.

Accordingly, alternate equations were developed, using the Nernstequation and first principles to develop a model which more closelysimulates the results. This was derived as follows. The Nernst equation,Equation 1 below, describes the ratio of oxidized (O) to reduced (R)molecules (number of molecules=n) at any given voltage and temperature,since not every molecule gets oxidized at the same oxidation potential.

$\begin{matrix}{{Equation}\mspace{14mu} 1} & \; \\{E_{DC} = {E_{0} + {\frac{RT}{nF}\ln\frac{\lbrack 0\rbrack}{\lbrack R\rbrack}}}} & (1)\end{matrix}$

E_(DC) is the electrode potential, E₀ is the formal potential of themetal complex, R is the gas constant, T is the temperature in degreesKelvin, n is the number of electrons transferred, F is faraday'sconstant, [O] is the concentration of oxidized molecules and [R] is theconcentration of reduced molecules.

The Nernst equation can be rearranged as shown in Equations 2 and 3:

$\begin{matrix}{{Equation}\mspace{14mu} 2} & \; \\{{E_{DC} - E_{0}} = {\frac{RT}{nF}\ln\frac{\lbrack 0\rbrack}{\lbrack R\rbrack}}} & (2)\end{matrix}$

E_(DC) is the DC component of the potential.

$\begin{matrix}{{Equation}\mspace{14mu} 3} & \; \\{\exp^{\frac{nF}{RT}{({E_{DC} - E_{0}})}} = \frac{\lbrack 0\rbrack}{\lbrack R\rbrack}} & (3)\end{matrix}$

Equation 3 can be rearranged as follows, using normalization of theconcentration to equal 1 for simplicity, as shown in Equations 4, 5 and6. This requires the subsequent multiplication by the total number ofmolecules.[O]+[R]=1  Equation 4[O]=1−[R]  Equation 5[R]=1−[O]  Equation 6

Plugging Equation 5 and 6 into Equation 3, and the fact that nF/RTequals 38.9 V⁻¹, for n=1, gives Equations 7 and 8, which define [O] and[R], respectively:

$\begin{matrix}{{Equation}\mspace{14mu} 7} & \; \\{\lbrack 0\rbrack = \frac{\exp^{38.9{({E - E_{0}})}}}{1 + \exp^{38.9{({E - E_{0}})}}}} & (4) \\{{Equation}\mspace{14mu} 8} & \; \\{\lbrack R\rbrack = \frac{1}{1 + \exp^{38.9{({E - E_{0}})}}}} & (5)\end{matrix}$

Taking into consideration the generation of an AC faradaic current, theratio of [O]/[R] at any given potential must be evaluated. At aparticular E_(DC) with an applied E_(AC), as is generally describedherein, at the apex of the E_(AC) more molecules will be in the oxidizedstate, since the voltage on the surface is now (E_(DC)+E_(AC)); at thebottom, more will be reduced since the voltage is lower. Therefore, theAC current at a given E_(DC) will be dictated by both the AC and DCvoltages, as well as the shape of the curve. Specifically, if the numberof oxidized molecules at the bottom of the AC cycle is subtracted fromthe amount at the top of the AC cycle, the total change in a given ACcycle is obtained, as is generally described by Equation 9. Dividing by2 then gives the AC amplitude.i _(AC)=(electrons at E _(DC) +E _(AC))−(electrons at E _(DC) −E_(AC))  Equation 9

Equation 10 thus describes the AC current which should result:i _(AC) =C ₀ Fω½([0]_(E) _(DC) _(+E) _(AC) −[0]_(E) _(DC) _(−E) _(AC))(6)  Equation 10

As depicted in Equation 11, the total AC current will be the number ofredox molecules C), times faraday's constant (F), times the AC frequency(ω), times 0.5 (to take into account the AC amplitude), times the ratiosderived above in Equation 7. The AC voltage is approximated by theaverage, E_(AC)2/π.

$\begin{matrix}{{{Equation}\mspace{14mu} 11}\mspace{605mu}} & \; \\{i_{AC} = {{\frac{C_{0}F\;\omega}{2}\left( \frac{\exp^{38.9{\lbrack{E_{DC} + \frac{E_{AC}2}{\pi} - E_{0}}\rbrack}}}{1 + \exp^{38.9{\lbrack{E_{DC} + \frac{E_{AC}2}{\pi} - E_{0}}\rbrack}}} \right)} - \frac{\exp^{38.9{\lbrack{E_{DC} + \frac{E_{AC}2}{\pi} - E_{0}}\rbrack}}}{1 + \exp^{38.9\lbrack{E_{DC} + \frac{E_{AC}2}{\pi} - E}}}}} & (7)\end{matrix}$

Using Equation 11, simulations were generated using increasingoverpotential. FIG. 22A shows one of these simulations, while FIG. 22Bdepicts a simulation based on traditional theory. FIGS. 23A and 23Bdepicts actual experimental data using the Fc-wire of Example 7 plottedwith the simulation, and shows that the model fits the experimental datavery well. In some cases the current is smaller than predicted, howeverthis has been shown to be caused by ferrocene degradation which may beremedied in a number of ways. However, Equation 11 does not incorporatethe effect of electron transfer rate nor of instrument factors. Electrontransfer rate is important when the rate is close to or lower than theapplied frequency. Thus, the true i_(AC) should be a function of allthree, as depicted in Equation 12.i _(AC) =f(Nernst factors)f(k _(ET))f(instrument factors)  Equation 12

These equations can be used to model and predict the expected ACcurrents in systems which use input signals comprising both AC and DCcomponents. As outlined above, traditional theory surprisingly does notmodel these systems at all, except for very low voltages.

In general, a single stranded probe nucleic acid system has a highimpedance, and a double stranded nucleic acid system (i.e. probehybridized to target to form a hybridization complex) has a lowerimpedance. This difference in impedance serves as the basis of a numberof useful AC detection techniques, as outlined below, but as will beappreciated by those in the art, a wide number of techniques may beused. In addition, the use of AC input and output signals enables theidentification of different species based on phase shifting between theAC voltage applied and the voltage or current response. Thus, ACdetection gives several advantages as is generally discussed below,including an increase in sensitivity, the ability to monitor changesusing phase shifting, and the ability to “filter out” background noise.

Accordingly, when using AC initiation and detection methods, thefrequency response of the system changes as a result of hybridization toform a double-stranded nucleic acid. By “frequency response” herein ismeant a modification of signals as a result of electron transfer betweenthe electrode and the second electron transfer moiety. This modificationis different depending on signal frequency. A frequency responseincludes AC currents at one or more frequencies, phase shifts, DC offsetvoltages, faradaic impedance, etc.

In a preferred embodiment, a target sequence is added to a probe singlestranded nucleic acid. Preferably, the probe single stranded nucleicacid comprises a covalently attached first electron transfer moietycomprising an electrode, and a covalently attached second electrontransfer moiety as described above. However, as outlined herein, it isalso possible to use a variety of other configurations in the system,including a second electron transfer moiety attached to the targetnucleic acid, a second probe nucleic acid containing a second electrontransfer moiety, intervening nucleic acids, etc.

In a preferred embodiment, the single stranded nucleic acid iscovalently attached to the electrode via a spacer. By “spacer” herein ismeant a moiety which holds the nucleic acid off the surface of theelectrode. In a preferred embodiment, the spacer is a conductiveoligomer as outlined herein, although suitable spacer moieties includepassayation agents and insulators as outlined above. The spacer moietiesmay be substantially non-conductive, although preferably (but notrequired) is that the rate of electron transfer through the spacer isfaster than the rate through single stranded nucleic acid, althoughsubstantially non-conductive spacers are generally preferred. Ingeneral, the length of the spacer is as outlined for conductive polymersand passayation agents. Similarly, spacer moieties are attached as isoutlined above for conductive oligomers, passayation agents andinsulators, for example using the same “A” linker defined herein.

The target sequence is added to the composition under conditions wherebythe target sequence, if present, will bind to the probe single strandednucleic acid to form a hybridization complex, as outlined above.

A first input electrical signal is then applied to the system,preferably via at least the sample electrode (containing the complexesof the invention) and the counter electrode, to initiate electrontransfer between the electrode and the second electron transfer moiety.Three electrode systems may also be used, with the voltage applied tothe reference and working electrodes. The first input signal comprisesat least an AC component. The AC component may be of variable amplitudeand frequency. Generally, for use in the present methods, the ACamplitude ranges from about 1 mV to about 1.1 V, with from about 10 mVto about 800 mV being preferred, and from about 10 mV to about 500 mVbeing especially preferred. The AC frequency ranges from about 0.01 Hzto about 10 MHz, with from about 1 Hz to about 1 MHz being preferred,and from about 1 Hz to about 100 kHz being especially preferred.

Surprisingly, the use of combinations of AC and DC signals allows thedifferentiation between single-stranded nucleic acid and double strandednucleic acid, as is outlined herein. In addition, signals comprised ofAC and DC components also allow surprising sensitivity and signalmaximization.

In a preferred embodiment, the first input signal comprises a DCcomponent and an AC component. That is, a DC offset voltage between thesample and counter electrodes is swept through the electrochemicalpotential of the second electron transfer moiety (for example, whenferrocene is used, the sweep is generally from 0 to 500 mV). The sweepis used to identify the DC voltage at which the maximum response of thesystem is seen. This is generally at or about the electrochemicalpotential of the second electron transfer moiety. Once this voltage isdetermined, either a sweep or one or more uniform DC offset voltages maybe used. DC offset voltages of from about −1 V to about +1.1 V arepreferred, with from about −500 mV to about +800 mV being especiallypreferred, and from about −300 mV to about 500 mV being particularlypreferred. In a preferred embodiment, the DC offset voltage is not zero.On top of the DC offset voltage, an AC signal component of variableamplitude and frequency is applied. If the nucleic acid has a low enoughimpedance to respond to the AC perturbation, an AC current will beproduced due to electron transfer between the electrode and the secondelectron transfer moiety.

For defined systems, it may be sufficient to apply a single input signalto differentiate between single stranded and double stranded (i.e. thepresence of the target sequence) nucleic acid. Alternatively, aplurality of input signals are applied. As outlined herein, this maytake a variety of forms, including using multiple frequencies, multipleDC offset voltages, or multiple AC amplitudes, or combinations of any orall of these.

Thus, in a preferred embodiment, multiple DC offset voltages are used,although as outlined above, DC voltage sweeps are preferred. This may bedone at a single frequency, or at two or more frequencies.

In a preferred embodiment, the AC amplitude is varied. Without beingbound by theory, it appears that increasing the amplitude increases thedriving force. Thus, higher amplitudes, which result in higheroverpotentials give faster rates of electron transfer. Thus, generally,the same system gives an improved response (i.e. higher output signals)at any single frequency through the use of higher overpotentials at thatfrequency. Thus, the amplitude may be increased at high frequencies toincrease the rate of electron transfer through the system, resulting ingreater sensitivity. In addition, this may be used, for example, toinduce responses in slower systems such as single stranded nucleic acidsfor identification, calibration and/or quantification. Thus, the amountof unhybridized single stranded nucleic acid on an electrode may becompared to the amount of hybridized double stranded nucleic acid toquantify the amount of target sequence in a sample. This is quitesignificant to serve as an internal control of the sensor or system.This allows a measurement either prior to the addition of target orafter, on the same molecules that will be used for detection, ratherthan rely on a similar but different control system. Thus, the actualmolecules that will be used for the detection can be quantified prior toany experiment. For example, a preliminary run at 1 Hz or less, forexample, will quantify the actual number of molecules that are on thesurface of the electrode. The sample can then be added, an output signaldetermined, and the ratio of bound/unbound molecules determined. This isa significant advantage over prior methods.

In a preferred embodiment, measurements of the system are taken at leasttwo separate amplitudes or overpotentials, with measurements at aplurality of amplitudes being preferred. As noted above, changes inresponse as a result of changes in amplitude may form the basis ofidentification, calibration and quantification of the system. Inaddition, one or more AC frequencies can be used as well.

In a preferred embodiment, the AC frequency is varied. At differentfrequencies, different molecules respond in different ways. As will beappreciated by those in the art, increasing the frequency generallyincreases the output current. However, when the frequency is greaterthan the rate at which electrons may travel between the electrode andthe second electron transfer moiety, higher frequencies result in a lossor decrease of output signal. For example, as depicted in FIG. 11, aresponse may be detected at 1 Hz for both single stranded nucleic acidand double stranded nucleic acid. However, at the higher frequencies,such as 200 Hz and above, the response of the single stranded nucleicacid is absent, while the response of the double stranded nucleic acidcontinues to increase. At some point, the frequency will be greater thanthe rate of electron transfer through even double-stranded nucleic acid,and then the output signal will also drop. Thus, the different frequencyresponses of single stranded and double stranded nucleic acids, based onthe rate at which electrons may travel through the nucleic acid (i.e.the impedance of the nucleic acid), forms the basis of selectivedetection of double stranded nucleic acids versus single strandednucleic acids.

In one embodiment, detection utilizes a single measurement of outputsignal at a single frequency. That is, the frequency response of asingle stranded nucleic acid can be previously determined to be very lowat a particular high frequency. Using this information, any response ata high frequency, for example such as 10 to 100 kHz, where the frequencyresponse of the single stranded nucleic acid is very low or absent, willshow the presence of the double stranded hybridization complex. That is,any response at a high frequency is characteristic of the hybridizationcomplex. Thus, it may only be necessary to use a single input highfrequency, and any frequency response is an indication that thehybridization complex is present, and thus that the target sequence ispresent.

In addition, the use of AC techniques allows the significant reductionof background signals at any single frequency due to entities other thanthe covalently attached nucleic acids, i.e. “locking out” or “filtering”unwanted signals. That is, the frequency response of a charge carrier orredox active molecule in solution will be limited by its diffusioncoefficient and charge transfer coefficient. Accordingly, at highfrequencies, a charge carrier may not diffuse rapidly enough to transferits charge to the electrode, and/or the charge transfer kinetics may notbe fast enough. This is particularly significant in embodiments that donot utilize a passayation layer monolayer or have partial orinsufficient monolayers, i.e. where the solvent is accessible to theelectrode. As outlined above, in DC techniques, the presence of “holes”where the electrode is accessible to the solvent can result in solventcharge carriers “short circuiting” the system. However, using thepresent AC techniques, one or more frequencies can be chosen thatprevent a frequency response of one or more charge carriers in solution,whether or not a monolayer is present. This is particularly significantsince many biological fluids such as blood contain significant amountsof redox active molecules which can interfere with amperometricdetection methods.

In a preferred embodiment, measurements of the system are taken at leasttwo separate frequencies, with measurements at a plurality offrequencies being preferred. A plurality of frequencies includes a scan.For example, measuring the output signal, e.g., the AC current, at a lowinput frequency such as 1-20 Hz, and comparing the response to theoutput signal at high frequency such as 10-100 kHz will show a frequencyresponse difference between double stranded nucleic acids with fastelectron transfer rates and single stranded nucleic acids with slowelectron transfer rates. In a preferred embodiment, the frequencyresponse is determined at least two, preferably at least about five, andmore preferably at least about ten frequencies.

After transmitting the input signal to initiate electron transfer, anoutput signal is received or detected. The presence and magnitude of theoutput signal will depend on the overpotential/amplitude of the inputsignal; the frequency of the input AC signal; the composition of theintervening medium, i.e. the impedance, between the electron transfermoieties (i.e. single stranded versus double stranded, etc.); the DCoffset; the environment of the system; the nature of the second electrontransfer moiety; and the solvent. At a given input signal, the presenceand magnitude of the output signal will depend in general on theimpedance of the medium between the two electron transfer moieties andthe character of the input signal. Double stranded nucleic acids, i.e.hybridization complexes, have relatively low impedance as compared tosingle stranded nucleic acids, and thus result in greater outputsignals. However, as noted herein, single stranded nucleic acids, in theabsence of the complementary target, can result in electron transferbetween the electron transfer moieties. Thus, upon transmitting theinput signal, comprising an AC component and a DC offset, electrons aretransferred between the first electron moiety, i.e. the electrode, andthe second electron moiety covalently attached to the nucleic acid, whenthe impedance is low enough, the frequency is in range, and theamplitude is sufficient, resulting in an output signal.

In a preferred embodiment, the output signal comprises an AC current. Asoutlined above, the magnitude of the output current will depend on anumber of parameters. By varying these parameters, the system may beoptimized in a number of ways.

In general, AC currents generated in the present invention range fromabout 1 femptoamp to about 1 milliamp, with currents from about 50femptoamps to about 100 microamps being preferred, and from about 1picoamp to about 1 microamp being especially preferred.

In a preferred embodiment, the output signal is phase shifted in the ACcomponent relative to the input signal. Without being bound by theory,it appears that surprisingly, the systems of the present invention aresufficiently uniform to allow phase-shifting based detection. That is,the complex biomolecules of the invention through which electrontransfer occurs react to the AC input in a homogeneous manner, similarto standard electronic components, such that a phase shift can bedetermined. This may serve as the basis of detection betweensingle-stranded and double stranded nucleic acids, but more importantly,may allow the detection of mismatches, since small changes in impedance,such as would be assumed from a mismatch present in the hybridizationcomplex, may effect the output AC phase in a greater manner than thefrequency response.

The output signal is characteristic of electron transfer through thehybridization complex; that is, the output signal is characteristic ofthe presence of double stranded nucleic acid. In a preferred embodiment,the basis of the detection is a difference in the faradaic impedance ofthe system as a result of the formation of the hybridization complex.Faradaic impedance is the impedance of the system between the twoelectron transfer moieties, i.e. between the electrode and the secondelectron transfer moiety. Faradaic impedance is quite different from thebulk or dielectric impedance, which is the impedance of the bulksolution between the electrodes. Many factors may change the faradaicimpedance which may not effect the bulk impedance, and vice versa. Thus,nucleic acids in this system have a certain faradaic impedance, thatwill depend on the distance between the electron transfer moieties,their electronic properties, and the composition of the interveningmedium, among other things. Of importance in the methods of theinvention is that the faradaic impedance between the electron transfermoieties is significantly different depending on whether the interveningnucleic acid is single stranded or double stranded. Thus, the faradaicimpedance of the system changes upon the formation of a hybridizationcomplex, and it is this change which is characteristic of thehybridization complex.

Accordingly, the present invention further provides apparatus for thedetection of nucleic acids using AC detection methods. The apparatusincludes a test chamber which has at least a first measuring or sampleelectrode, and a second measuring or counter electrode. Three electrodesystems are also useful. The first and second measuring electrodes arein contact with a test sample receiving region, such that in thepresence of a liquid test sample, the two electrodes may be inelectrical contact.

In a preferred embodiment, the first measuring electrode comprises asingle stranded nucleic acid covalently attached via a spacer, andpreferably via a conductive oligomer, such as are described herein. Inone embodiment, the second electron transfer moiety may be attached tothe probe single stranded nucleic acid, or it may be attached to asecond probe nucleic acid, the target nucleic acid, or may be addedseparately, for example as an intercalator. In a preferred embodiment,the second electron transfer moiety is covalently attached to the probesingle stranded nucleic acid.

The apparatus further comprises an AC voltage source electricallyconnected to the test chamber; that is, to the measuring electrodes.Preferably, the AC voltage source is capable of delivering DC offsetvoltage as well.

In a preferred embodiment, the apparatus further comprises a processorcapable of comparing the input signal and the output signal. Theprocessor is coupled to the electrodes and configured to receive anoutput signal, and thus detect the presence of the target nucleic acid.

Thus, the compositions of the present invention may be used in a varietyof research, clinical, quality control, or field testing settings.

In a preferred embodiment, the probes are used in genetic diagnosis. Forexample, probes can be made using the techniques disclosed herein todetect target sequences such as the gene for nonpolyposis colon cancer,the BRCA1 breast cancer gene, P53, which is a gene associated with avariety of cancers, the Apo E4 gene that indicates a greater risk ofAlzheimer's disease, allowing for easy presymptomatic screening ofpatients, mutations in the cystic fibrosis gene, or any of the otherswell known in the art.

In an additional embodiment, viral and bacterial detection is done usingthe complexes of the invention. In this embodiment, probes are designedto detect target sequences from a variety of bacteria and viruses. Forexample, current blood-screening techniques rely on the detection ofanti-HIV antibodies. The methods disclosed herein allow for directscreening of clinical samples to detect HIV nucleic acid sequences,particularly highly conserved HIV sequences. In addition, this allowsdirect monitoring of circulating virus within a patient as an improvedmethod of assessing the efficacy of anti-viral therapies. Similarly,viruses associated with leukemia, HTLV-I and HTLV-II, may be detected inthis way. Bacterial infections such as tuberculosis, chlamydia and othersexually transmitted diseases, may also be detected.

In a preferred embodiment, the nucleic acids of the invention find useas probes for toxic bacteria in the screening of water and food samples.For example, samples may be treated to lyse the bacteria to release itsnucleic acid, and then probes designed to recognize bacterial strains,including, but not limited to, such pathogenic strains as, Salmonella,Campylobacter, Vibrio cholerae, enterotoxic strains of E. coli, andLegionnaire's disease bacteria. Similarly, bioremediation strategies maybe evaluated using the compositions of the invention.

In a further embodiment, the probes are used for forensic “DNAfingerprinting” to match crime-scene DNA against samples taken fromvictims and suspects.

In an additional embodiment, the probes in an array are used forsequencing by hybridization.

The present invention also finds use as a unique methodology for thedetection of mutations or mismatches in target nucleic acid sequences.As a result, if a single stranded nucleic acid containing electrontransfer moieties is hybridized to a target sequence with a mutation,the resulting perturbation of the base pairing of the nucleosides willmeasurably affect the electron transfer rate. This is the case if themutation is a substitution, insertion or deletion. Alternatively, twosingle stranded nucleic acids each with a covalently attached electrontransfer species that hybridize adjacently to a target sequence may beused. Accordingly, the present invention provides for the detection ofmutations in target sequences.

Thus, the present invention provides for extremely specific andsensitive probes, which may, in some embodiments, detect targetsequences without removal of unhybridized probe. This will be useful inthe generation of automated gene probe assays.

In an alternate embodiment the electron transfer moieties are onseparate strands. In this embodiment, one single stranded nucleic acidhas an electrode covalently attached via a conductive oligomer. Theputative target sequences are labelled with a second electron transfermoiety as is generally described herein, i.e. by incorporating anelectron transfer moiety to individual nucleosides of a PCR reactionpool. Upon hybridization of the two single-stranded nucleic acids,electron transfer is detected.

Alternatively, the compositions of the invention are useful to detectsuccessful gene amplification in PCR, thus allowing successful PCRreactions to be an indication of the presence or absence of a targetsequence. PCR may be used in this manner in several ways. For example,in one embodiment, the PCR reaction is done as is known in the art, andthen added to a composition of the invention comprising the targetnucleic acid with a second ETM, covalently attached to an electrode viaa conductive oligomer with subsequent detection of the target sequence.Alternatively, PCR is done using nucleotides labelled with a second ETM,either in the presence of, or with subsequent addition to, an electrodewith a conductive oligomer and a target nucleic acid. Binding of the PCRproduct containing second ETMs to the electrode composition will allowdetection via electron transfer. Finally, the nucleic acid attached tothe electrode via a conductive polymer may be one PCR primer, withaddition of a second primer labelled with an ETM. Elongation results indouble stranded nucleic acid with a second ETM and electrode covalentlyattached. In this way, the present invention is used for PCR detectionof target sequences.

In an additional embodiment, the present invention provides novelcompositions comprising metallocenes covalently attached via conductiveoligomers to an electrode, such as are generally depicted in Structure35:

Structure 35 utilizes a Structure 4 conductive oligomer, although aswill be appreciated by those in the art, other conductive oligomers suchas Structures 2, 3, 9 or 10 types may be used. Preferred embodiments ofStructure 35 are depicted below.

Preferred R groups of Structure 37 are hydrogen.

These compositions are synthesized as follows. The conductive oligomerlinked to the metallocene is made as described herein; see also, Hsunget al., Organometallics 14:4808-4815 (1995); and Bumm et al., Science271:1705 (1996), both of which are expressly incorporated herein byreference. The conductive oligomer is then attached to the electrodeusing the novel ethylpyridine protecting group, as outlined herein.

Once made, these compositions have unique utility in a number ofapplications, including photovoltaics, and infrared detection. Apreferred embodiment utilizes these compounds in calibrating apotentiostat, serving as an internal electrochemistry reference in anarray of the invention.

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.All references cited herein are incorporated by reference.

EXAMPLES Example 1 Synthesis of Conductive Oligomer Linked Via an Amideto a Nucleoside

This synthesis is depicted in FIG. 1, using uridine as the nucleosideand a Structure 4 phenyl-acetylene conductive oligomer.

Compound #1: To a solution of 10.0 gm (40 mmol) of 4-iodothioanisole in350 mL of dichloromethane cooled in an ice-water bath was added 10.1 gmof mCPBA. The reaction mixture was stirred for half hour and thesuspension was formed. To the suspension was added 4.0 gm of poweredCa(OH)₂, the mixture was stirred at room temperature for 15 min andfiltered off and the solid was washed once with 30 mL ofdichloromethane. To the combined filtrate was added 12 mL oftrifluoroacetic anhydride and the reaction mixture was refluxed for 1.5h under Argon. After removing the solvents, the residue was dissolved in200 mL of a mixture of TEA and methanol (ratio=50:50) and concentratedto dryness. The residue was dissolved in 100 mL of dichloromethane andthe solution was washed once with 60 mL of the saturated ammonimchloride solution. The aqueous layer was extracted twice withdichloromethane (2×70 mL). The organic extracts were combined and driedover anhydrous sodium sulfate and immediately concentrated to dryness asquickly as possible. The residue was dissolved in 120 mL of benzene,followed by adding 5.3 mL of 4-vinylpyridine. The reaction mixture wasrefluxed under Argon overnight. The solvent was removed and the residuewas dissolved in dichloromethane for column chromatography. Silica gel(150 gm) was packed with 20% ethyl acetate/hexane mixture. The crudeproduct solution was loaded and the column was eluted with 20 to 60%ethyl acetate/hexane mixture. The fractions was identified by TLC(EtOAc:Hexane=50:50, Rf=0.24) and pooled and concentrated to dryness toafford 7.4 gm (54.2%) of the solid title compound.

Compound #2: To a solution of 3.4 gm (9.97 mmol) of Compound #1 in 70 mLof diethylamine was added 200 mg of bis(triphenylphosphine)palladium(II) chloride, 100 mg of cuprous iodide and 1.9 mL oftrimethylsilylacetylene under Argon. The reaction mixture was stirredfor 2 h. After removing the diethylamine, the residue was dissolved indichloromethane for column chromatography. Silica gel (120 gm) waspacked with a cosolvent of 50% ethyl acetate/50% hexane. The crudesample solution was loaded and the column was eluted with the samecosolvent. After removing the solvents, the liquid title compound (2.6gm, 83.7%) was obtained.

Compound #3: To a solution of 2.6 gm of Compound #2 in 150 mL ofdichloromethane cooled in an ice-water bath was added 9.0 mL of 1 Ntetrabutylammonium fluoride THF solution. The reaction mixture wasstirred for 1 h. and washed once with water and dried over anhydrousNa₂SO₄. After removing the solvent, the residue was used for columnseparation. Silica gel (50 gm) was packed with a cosolvent of 50% ethylacetate 150% hexane. The crude product solution was loaded and thecolumn was eluted with the same solvents. The removal of the solventsgave the solid title compound (1.87 gm, 94.1%).

Compound #4: To a glass bottle were added 1.80 gm (7.52 mmol) ofCompound #3, 160 mg of bis(triphenylphosphine)palladium (II) chloride,80 mg of cuprous iodide and 2.70 gm (9.0 mmol) of1-trimethylsilyl-2-(4-iodophenyl)acetylene. The bottle was sealed andbubbled with Argon. Diethylamine was introduced by a syringe. Thereaction mixture was heated at 50° C. under Argon for 1 h. The amine wasremoved and the residue was dissolved in dichloromethane for theseparation. Silica gel (100 gm) was packed with 60% ethylacetate/hexane. The crude mixture was loaded and the column was elutedwith the same solvents. The fractions were identified by TLC(EtOAc:Hexane=50:50, the product emitted blue light) and pooled. Theremoval of the solvents gave the solid title product (2.47 gm, 79.8%).

Compound #5: To a solution of 2.47 gm of Compound #4 in 130 mL ofdichloromethane cooled in an ice-water bath was added 8.0 mL of 1 Ntetrabutylammonium fluoride THF solution. The reaction mixture wasstirred for 1 h. and washed once with water and dried over anhydrousNa₂SO₄. After removing the solvent, the residue was used for columnseparation. Silica gel (60 gm) was packed with a cosolvent of 50% ethylacetate/50% CH₂Cl₂. The crude solution was loaded and the column waseluted with the same solvents. The removal of solvents gave the solidtitle product (1.95 gm, 95.7%).

Compound #6: To a glass bottle were added 0.23 gm (0.68 mmol) ofCompound #5, 0.5 gm (0.64 mmol) of 2′-deoxy-2′-(4-iodophenylcarbonyl)amino-5′-O-DMT uridine, 60 mg of bis(triphenylphosphine)palladium (II)chloride, 30 mg of cuprous iodide. The bottle was sealed and bubbledwith Argon. Pyrrodine (15 mL) and DMF (15 mL) were introduced by asyringe. The reaction mixture was heated at 85° C. overnight. Thesolvents were removed in vacuo and the residue was dissolved in 300 mLof dichloromethane. The solution was washed three times with water anddried over sodium sulfate. After removing the solvent, the residue wassubjected to column purification. Silica gel (30 gm) was packed with 1%TEA/1% methanol/CH₂Cl₂ and the sample solution was loaded. The columnwas eluted with 1% TEA/1% methanol/CH₂Cl₂ and 1% TEA/2% methanol/CH₂Cl₂.The fractions were identified and concentrated to dryness. The separatedproduct was subjected to another reverse-phase column purification.Reverse-phase silica gel (C-18, 120 gm) was packed with 60% CH₃CN/40%H₂O and the sample was dissolved in very small amount of THF and loaded.The column was eluted with 100 mL of 60% CH₃CN/40% H₂O, 100 mL of 70%CH₃CN/30% H₂O, 100 mL of 60% CH₃CN/10% THF/30% H₂O, 200 mL of 50%CH₃CN/20% THF/30% H₂O and 500 mL of 35% CH₃CN/35% THF/30% H₂O. Thefractions were identified by HPLC (0.1 mM TEAA: CH3CN=20:80, flowrate=1.0 mL/min). and concentrated to dryness to afford a pure titlecompound.

Compound #7: To a solution of 100 mg (0.1 mmol) of pure compound # 6 in40 mL of pyridine were added 50 mgm of DMAP and 1.0 gm (10 mmol) ofsuccinic anhydride. The reaction mixture was stirred under Argon for 40h. After removing pyridine, the residue was dissolved in 300 mL ofdichloromethane, followed by adding 150 mL of 5% aqueous NaHCO₃solution. The mixture was vigorously stirred for 3 h and separated. Theorganic layer was washed once with 1% citric acid solution and driedover anhydrous sodium sulfate and concentrated to dryness to give 110mgm of Compound #7. Without further purification, the Compound #7 wasused for the preparation of the corresponding CPG.

Conductive oligomer-Uridine-CPG: To 1.4 gm of LCAA-CPG (500) in 100 mLround bottom flask were added 110 mgm (101 μmol) of the Compound #7, 100mgm (230 μmol) of BOP reagent, 30 mgm (220 μmol) of HBT, 70 mL ofdichloromethane and 2 mL of TEA. The mixture was shaken for three days.The CPG was filtered off and washed twice with dichloromethane andtransferred into another 100 mL flask. Into CPG were added 50 mL ofpyridine, 10 mL of acetic anhydride and 2 mL of N-methylimidazole. TheCPG was filtered off, washed twice with pyridine, methanol,dichloromethane and ether, and dried over a vacuum. The loading of thenucleoside was measured according to the standard procedure to be 7.1μmol/gm.

2′-Deoxy-2′-(4-iodophenylcarbonyl)amino-5′-O-DMT uridine: To a solutionof 5.1 gm (9.35 mmol) of 2′-deoxy-2′-amino-5′-O-DMT uridine in 250 mL ofpyridine cooled in an ice-water bath was added 3 mL ofchlorotrimethylsilane. The reaction mixture was warmed up to roomtemperature and stirred for 1 h. To the prepared solution were added 0.1gm of DMAP and 3.0 gm (10.9 mmol) 4-iodobezoyl chloride and the reactionmixture was stirred overnight. To this solution was added 30 mL ofconcentrated ammonium hydroxide solution and the mixture was stirred forexact 15 min. The solvents were removed in vacuo. The residue wasdissolved in 15 mL of dichloromethane for column separation. Silica gel(125 gm) was packed with 1% TEA/2% CH₃OH/CH₂Cl₂. After loading thesample, the column was eluted with 300 mL of 1% TEA/2% CH₃OH/CH₂Cl₂, and500 mL of 1% TEA/4% CH₃OH/CH₂Cl₂. The fractions were identified by TLC(CH₃OH:CH₂Cl₂=10:90) and pooled and concentrated to dryness to give 6.2gm (85.5%) of the pure title compound.

Synthesis of the Phosphoramidite (Compound # 8).

To a solution of 0.2 gm of Compound #6 and 30 mg of diisopropylammoniumtetrazolide in 10 mL of dry dichloromethane is added 0.12 gm of2-cyanoethyl N,N, N′,N′-tetraisopropylphosphane under Argon. Thesolution was stirred for 5 h and diluted by adding 60 mL ofdichloromethane. The solution was washed twice with 2.5% w/v sodiumbicarbonate solution, once with the brine and dried over sodium sulfate.After removing the solvent, residue was dissolved in 5 mL ofdichloromethane, followed by adding slowly 100 mL of hexane. Thesuspension was stored at −20° C. for 1 h. The supernatant was decantedand the residue was dried over a high vacuum overnight to afford 0.19 gm(79.0%) of the title product, which will be used for DNA synthesis.

Example 2 Synthesis of Conductive Oligomers Linked to the Ribose of aNucleoside Via an Amine Linkage Example 2A

Synthesis of 2′-(4-iodophenyl)amino-2′-deoxy-5′-O-DMT-uridine (Product4): This synthesis is depicted in FIG. 2, and reference is made to thelabelling of the products on the figure. To a solution of 5.0 gm of5′-O-DMT-uridine (Product 1) and 2.7 gm of dimethylaminopyridine in 200mL of acetonitrile was added 3.3 gm of p-iodophenyl isocyalidedichloride portion by portion under Argon. The reaction mixture wasstirred overnight. The mixture was diluted by adding 550 mL ofdichloromethane and washed twice with 5% sodium bicarbonate aqueoussolution and once with the brine solution, and then dried over sodiumsulfate. The removal of the solvent in vacuo gave the crude Product 2.Without further purification, Product 2 was dissolved in 50 mL of dryDMF and the solution was heated at 150° C. foe 2 h. After distillationof DMF, the residue was dissolved in 300 mL of dichloromethane, washedonce with 5% sodium bicarbonate solution, once with the brine solutionand dried over sodium sulfate. The removal of the solvent gave the crudeProduct 3. Without purification, the Product 3 was dissolved 100 mL of amixture of 50% Dioxane and 50% Methanol. To this solution was added 43mL of 1N NaOH solution. The reaction mixture was stirred overnight. Themixture was diluted by adding 800 mL of dichloromethane and washed twicewater and dried over Na₂SO₄. After removing the solvent, the residue wasdissolved in 15 mL of dichloromethane for the column separation. Silicagel (100 gm) of packed with 1% TEA/2% Ethanol/CH₂Cl₂, after loading thesample solution, the column was eluted with 1% TEA/2-3% Ethanol/CH₂Cl₂.The fractions were identified by TLC (CH3OH:CH2Cl2=1:9) and pooled andconcentrated to give 2.0 gm (29.2%) of the Product 4.

Additional conductive oligomer units can then be added to product 4 asoutlined herein, with additional nucleotides added and attachment to anelectrode surface as described herein.

Example 2B

Benzylamino-uridine was synthesized as shown in FIG. 16.

Synthesis of Compound C2: To a solution of 8.3 gm (15.7 mmol) ofcyclonucleoside C1 in 200 mL of dichloromethane was added 2.80 gm ofcarbonyldiimidazole under Argon. After the solution was stirred for 7 h,into this solution were added 4.3 gm of 4-iodobenzylamine and 10 mL ofdiisopropylethylamine. The mixture was stirred overnight under Argonatmosphere. The solution was washed twice with 5% Citric acid solutionand dried over sodium sulfate. After concentration, the residue wasdissolved in a small amount of dichloromethane for the columnseparation. Silica gel (150 gm) was packed with 1% TEA/2% CH₃OH/CH₂Cl₂,upon loading the sample solution, the column was eluted with 1%TEA/2-10% CH₃OH/CH₂Cl₂. The fractions were identified by TLC(CH₃OH:CH₂Cl₂ 7:93) and pooled and concentrated to afford 9.75 gm(78.8%) of the product C2.

Synthesis of Compound C3: A mixture of 9.75 gm (12.4 mmol) of thecompound C2 and 1.0 mL of DBU in 250 mL of dry THF was stirred at 50° C.under Argon for two days. THF was removed by a rotavapor and the residuewas dissolved 20 mL of dichloromethane for the purification. Silica gel(130 gm) was packed with 1% TEA/25% EtOAc/CH₂Cl₂, after loading thesample solution, the column was eluted with same solvent mixture. Thefractions containing the desired product was pooled and concentrated togive 6.46 gm (66.3%) of the product C3.

Synthesis of the Final Compound C4: The compound C3 (6.46 gm) wasdissolved in a mixture of 150 mL of 1,4-dioxane and 100 mL of methanol,followed by adding 100 mL of 4.0 M aqueous sodium hydroxide. The mixturewas stirred at room temperature overnight. The solution was diluted byadding 500 mL of dichloromethane and 500 mL of the brine solution. Themixture was shaken well and the organic layer was separated and washedonce with the 500 mL of the brine solution and dried over sodiumsulfate. The dichloromethane was removed by a rotavapor and the dioxanewas removed by a high vacuum. The residue was dissolved in 20 mL ofdichloromethane for the separation. Silica gel (80 gm) was packed with1% TEA/25% EtOAc/CH₂Cl₂ and the sample solution was loaded. The columnwas eluted with 1% TEA/25-50% EtOAc/CH₂Cl₂. The right fractions werecombined and concentrated to give 4.1 gm (65.7%) of the final productC4.

Example 3 Synthesis of a Conductive Oligomer with an R Group Attached tothe Y Aromatic Group

This synthesis is depicted in FIG. 6.

Synthesis of 2-Acetyl-5-iodotoluene (P 1). To a suspension of 20 gm ofaluminum trichloride in 500 mL of dichloromethane was added 10.2 mL ofacetyl chloride under Argon. After the reaction mixture was stirred for15 min, 3-iodotoluene (20 gm) was added through a syringe. The mixturewas stirred overnight under Argon and poured into 500 gm of ice-water.Organic layer was separated and washed once with the saturated ammoniumchloride solution, and washed once with 10% sodium thiosulfate solutionand dried over sodium sulfate. After removing the solvent, the residuewas dissolved in hexane for the column purification. Silica gel (260 gm)was packed with hexane, after loading the sample solution, the columnwas eluted with 750 mL of hexane, 750 mL of 1% v/v ether/hexane, 750 mLof 2% v/v ether/hexane and 1500 mL of 3% v/v ether/hexane. The fractionscontaining the right isomer were identified by GC-MS and ¹H NMR andpooled and concentrated to dryness to afford 12.2 gm (51.2%) of thetitle product (P 1).

Iodo-3-methyl-4-(ehynyl trimethylsilyl)benzene (P2). Under inertatmosphere 500 ml bound bottom flask was charged with 25 ml of dry THF,cooled to −78° C. and 14 ml of 2.0 M LDA solution(heptane/ethylbenzene/THF solution) was added by syringe. To thissolution 6.34 gr (24.38 mmole) of iodo-3-methyl-4-acetyl benzene in 25ml of THF was added dropwise and the reaction mixture was stirred for 1hr at −78° C., then 4.0 ml (19.42 mmole) of diethychlorophosphate wereadded by syringe. After 15 min cooling bath was removed and the reactionmixture was allowed to heat up to RT and stirred for 3 hrs. The resultedmixture was cooled again to −78° C. and 29 ml of 2.0 M LDA solution wereadded dropwise. At the end of the addition the reaction mixture wasallowed to warm up to RT and stirred for additional 3 hrs. After thatperiod of time it was cooled again to −20° C., 9.0 ml (70.91 mmole) oftrimethylsilyl chloride were injected and the stirring was continued for2 hrs at RT. The reaction mixture was poured into 200 ml of ice/sodiumbicarbonate saturated aqueous solution and 300 ml of ether were added toextract organic compounds. The aqueous phase was separated and extractedagain with 2×100 ml of ether. The ether fractions were combined, driedover sodium sulfate and evaporated. The resulted liquid residue waspurified by silica gel chromatography (100% n-hexane as eluent). 4.1 gr(54% yield) were obtained.

Synthesis of Product (P 3). To a solution of 1.14 gm of Compound #3 (asdescribed above) and 1.60 gm of P 2 in 100 mL of diethylamine were added0.23 gm of [1,1′-bis(diphenylphosphino)ferrocene]palladium (II) chlorideand 0.1 gm of copper (I) iodide under Argon. The reaction mixture wasstirred at 55° C. for 1 h and stirred at room temperature overnight.After removing the solvent, the residue was dissolved in dichloromethanefor column separation. Silica gel (120 gm) was packed with 20% ethylacetate/CH₂Cl₂. The sample solution was loaded and the column was elutedwith 20-50% ethyl acetate/CH₂Cl₂. The fractions were identified by TLC(EtOAC: CH2Cl2=50: 50) and pooled and concentrated to give 1.70 gm(84.0%) of TMS-derivative of P 3.

To a solution of 0.74 gm of TMS-derivative of P 3 in 70 mL ofdichloromethane at 0° C. was added 2.2 mL of 1.0 M (nBu)₄NF THFsolution. After stirring for 30 min, the solution was washed once withwater and dried over sodium sulfate. The solvent was removed, theresidue was used for column separation. Silica gel (20 gm) was packedwith 20% ethyl acetate/CH₂Cl₂, the column was eluted with 20-40% ethylacetate/CH₂Cl₂. The fractions containing the fluorescent compound werecombined and concentrated to dryness to afford 0.5 gm (81.3%) of thepure P 3.

Synthesis of P 4: To a solution of 0.5 gm of P 3 and 0.63 gm of P 2 in50 mL of dry DMF and 10 mL of TEA were added 100 mgm of[1,1′-bis(diphenylphosphino)ferrocene]palladium (II) chloride and 50 mgmof copper (I) iodide under Argon. The reaction mixture was stirred at55° C. for 1 h and stirred at 35° C. overnight. The solvents wereremoved in vacuo and the residue was dissolved in 10 mL of CH₂Cl₂ forcolumn separation. Silica gel (100 gm) was packed with 20% ethylacetate/CH₂Cl₂, after loading the sample, the column was eluted with20-40% ethyl acetate/CH₂Cl₂. The fractions were identified by TLC(EtOAC:CH₂Cl₂=50:50) and pooled and concentrated to give 0.47 gm (61.3%)of TMS-derivative of P 4.

To a solution of 0.47 gm of TMS-derivative of P 4 in 70 mL ofdichloromethane at 0° C. was added 1.0 mL of 1.0 M (nBu)₄NF THFsolution. After stirring for 30 min, the solution was washed once withwater and dried over sodium sulfate. The solvent was removed, theresidue was used for column separation. Silica gel (20 gm) was packedwith 20% ethyl acetate/CH₂Cl₂, the column was eluted with 20-40% ethylacetate/CH₂Cl₂. The fractions containing the fluorescent compound werecombined and concentrated to dryness to afford 0.32 gm (78.7%) of thepure P 4.

Other conductive oligomers with R groups are depicted in FIG. 17, whichwere made using the techniques outlined herein.

Example 4 Synthesis of a Nucleoside with a Metallocene Second ElectronTransfer Moiety Attached Via a Ribose

Synthesis of 5′-O-DMT-2′-deoxy-2′-(ferrocenecarbonyl)amino Uridine(UAF): To a solution of 2.5 gm (10.9 mmol) of ferrocene monocarboxylicacid in 350 mL of dichloromethane were added 2.25 gm (10.9 mmol) of DCCand 1.27 gm (10.9 mmol) of N-hydroxysuccinimide. The reaction mixturewas stirred for 3 h and the precipitate was formed. The precipitate wasfiltered off and washed once with dichloromethane. The combined filtratewas added into 4.5 gm (8.25 mmol) of 2′-deoxy-2′-amino-5′-O-DMT uridine,followed by adding 2 mL of triethylamine. The reaction mixture wasstirred at room temperature for 8 days. After removing the solvent, theresidue was dissolved in dichloromethane for separation. Silica gel (120gm) was packed with 1% TEA/2% CH₃OH/CH₂Cl₂. After loading the samplesolution, the column was eluted with 2-7% CH₃OH/1% TEA/CH₂Cl₂. Thefraction was identified by TLC(CH₃OH:CH₂Cl₂=1:9) and pooled andconcentrated to dryness to afford 1.3 gm (22.0%) of the title compound.

Synthesis of UAF Phosphoramidite:

Preparation of Diisopropylaminochloro(β-cyano)ethoxyphosphine: To asolution of 0.54 mL (4.0 mmol) of dichloro(β-cyano)ethoxyphosphine in 40mL of dichloromethane cooled in an ice-water bath was added 10 mL ofdiisopropylethylamine, followed by adding 0.64 mL (4.0 mmol) ofdiisopropylamine under Argon. The reaction mixture was warmed up to roomtemperature and stirred for 2 h. After adding 0.1 gm of DMAP into thesolution, the reaction mixture is ready for the next step reaction.

Preparation of UAF phosphoramidite: To a solution of 1.30 gm (1.72 mmol)of 5′-O-DMT-5-ferrocenylacetylenyl-2′-deoxy uridine in 40 mL ofdichloromethane cooled in an ice-water bath was added 10 mL ofdiisopropylethylamine. The prepared phosphine solution was transferredinto the nucleoside solution through a syringe. The reaction mixture waswarmed up to room temperature and stirred overnight. The solution wasdiluted by adding 100 mL of dichloromethane and washed once with 200 mLod 5% aqueous NaHCO₃ solution, and once with the brine (200 mL) anddried over Na₂SO₄ and concentrated to dryness. Silica gel (47 gm) waspacked with 2% TEA/1% CH₃OH/CH₂Cl₂. The residue was dissolved in 10 mLof dichloromethane and loaded. The column was eluted with 150 mL of 1%TEA/1% CH₃OH/CH₂Cl₂ and 250 mL of 1% TEA/2% CH₃OH/CH₂Cl₂. The fractionswere pooled and concentrated to give 0.5 gm (30.3%) of the titlecompound.

Nucleotides containing conductive oligomers and second electron transfermoieties were incorporated into nucleic acids using standard nucleicacid synthesis techniques; see “Oligonucleotides and Analogs, APractical Approach”, Ed. By F. Eckstein, Oxford University Press, 1991,hereby incorporated by reference.

Example 5 Synthesis of a Nucleoside with a Metallocene Second ElectronTransfer Moiety Attached Via the Base

Synthesis of 5′-O-DMT-5-ferrocenylacetylenyl-2′-deoxy uridine (UBF): Ina flask were added 4.8 gm (13.6 mmol) of 5-iodo-2′-deoxy uridine, 400 mgof bis(triphenylphosphine)palladium (II) chloride, 100 mg of cuprousiodide, 95 mL of DMF and 10 mL of TEA. The solution was degassed byArgon and the flask was sealed. The reaction mixture was stirred at 50°C. overnight. After removing solvents in vacuo, the residue wasdissolved in 140 mL of dry pyridine, followed by adding 0.2 gm of DMAPand 5.0 gm (14.8 mmol) of DMT-Cl. The reaction mixture was stirred at RTovernight. After removing the solvent, the residue was dissolved in 300mL of dichlormethane and washed twice with 5% aqueous NaHCO₃ (2×200 mL),twice with the brine (2×200 mL) and dried over sodium sulfate. Thesolvent was removed and the residue was coevaporated twice with tolueneand dissolved in 15 mL of dichloromethane for column separation. Silicagel (264 gm) was packed 0.5% TEA/CH₂Cl₂. After loading the crude productsolution, the column was eluted with 300 mL of 1% TEA/2% CH₃OH/CH₂Cl₂,400 mL of 1% TEA/5% CH₃OH/CH₂Cl₂, and 1.2 L of 1% TEA/7% CH₃OH/CH₂Cl₂.The fractions were identified by TLC(CH₃OH:CH₂Cl₂=10:90) and pooled andconcentrated to dryness to give 7.16 gm (71.3%) of the title compound.

Synthesis of UBF Phosphoramidite:

Preparation of Diisopropylaminochloro(β-cyano)ethoxyphosphine: To asolution of 1.9 mL (13.8 mmol) of dichloro(β-cyano)ethoxyphosphine in 40mL of dichloromethane cooled in an ice-water bath was added 10 mL ofdiisopropylethylamine, followed by adding 2.3 mL (13.8 mmol) ofdiisopropylamine under Argon. The reaction b mixture was warmed up toroom temperature and stirred for 2 h. After adding 0.1 gm of DMAP intothe solution, the reaction mixture is ready for next step reaction.

Preparation of UBF phosphoramidite: To a solution of 3.42 gm (4.63 mmol)of 5′-O-DMT-5-ferrocenylacetylenyl-2′-deoxy uridine in 40 mL ofdichloromethane cooled in an ice-water bath was added 10 mL ofdiisopropylethylamine. The prepared phosphine solution was transferredinto the nucleoside solution through a syringe. The reaction mixture waswarmed up to room temperature and stirred overnight. The solution wasdiluted by adding 150 mL of dichloromethane and washed once with 200 mLof 5% aqueous NaHCO₃ solution, and once with the brine (200 mL) anddried over Na₂SO₄ and concentrated to dryness. Silica gel (92 gm) waspacked with 2% TEA/1% CH₃OH/CH₂Cl₂. The residue was dissolved in 10 mLof dichloromethane and loaded. The column was eluted with 500 mL of 1%TEA/2% CH₃OH/CH₂Cl₂. The fractions were pooled and concentrated to give3.0 gm (69.0%) of the title compound.

Nucleotides containing conductive oligomers and second electron transfermoieties were incorporated into nucleic acids using standard nucleicacid synthesis techniques; see “Oligonucleotides and Analogs, APractical Approach”, Ed. By F. Eckstein, Oxford University Press, 1991,hereby incorporated by reference.

Example 6 Synthesis of an Electrode Containing Nucleic Acids ContainingConductive Oligomers with a Monolayer of (CH₂)₁₆

Using the above techniques, and standard nucleic acid synthesis, theuridine with the phenyl-acetylene conductive polymer of Example 1 wasincorporated at the 3′ position to form the following nucleic acid:ACCATGGACTCAGCU(SEQ ID NO: 1)-conductive polymer of Example 1(hereinafter “wire-1”).

HS—(CH2)16-OH (herein “insulator-2”) was made as follows.

16-Bromohexadecanoic acid. 16-Bromohexadecanoic acid was prepared byrefluxing for 48 hrs 5.0 gr (18.35 mmole) of 16-hydroxyhexadecanoic acidin 24 ml of 1:1 v/v mixture of HBr (48% aqueous solution) and glacialacetic acid. Upon cooling, crude product was solidified inside thereaction vessel. It was filtered out and washed with 3×100 ml of coldwater. Material was purified by recrystallization from n-hexane,filtered out and dried on high vacuum. 6.1 gr (99% yield) of the desiredproduct were obtained.

16-Mercaptohexadecanoic acid. Under inert atmosphere 2.0 gr of sodiummetal suspension (40% in mineral oil) were slowly added to 100 ml of drymethanol at 0° C. At the end of the addition reaction mixture wasstirred for 10 min at RT and 1.75 ml (21.58 mmole) of thiolacetic acidwere added. After additional 10 min of stirring, 30 ml degassedmethanolic solution of 6.1 gr (18.19 mmole) of 16-bromohexadecanoic acidwere added. The resulted mixture was refluxed for 15 hrs, after which,allowed to cool to RT and 50 ml of degassed 1.0 M NaOH aqueous solutionwere injected. Additional refluxing for 3 hrs required for reactioncompletion. Resulted reaction mixture was cooled with ice bath andpoured, with stirring, into a vessel containing 200 ml of ice water.This mixture was titrated to pH=7 by 1.0 M HCl and extracted with 300 mlof ether. The organic layer was separated, washed with 3×150 ml ofwater, 150 ml of saturated NaCl aqueous solution and dried over sodiumsulfate. After removal of ether material was purified byrecrystallization from n-hexane, filtering out and drying over highvacuum. 5.1 gr (97% yield) of the desired product were obtained.

16-Bromohexadecan-1-ol. Under inert atmosphere 10 ml of BH₃.THF complex(1.0 M THF solution) were added to 30 ml THF solution of 2.15 gr (6.41mmole) of 16-bromohexadecanoic acid at −20° C. Reaction mixture wasstirred at this temperature for 2 hrs and then additional 1 hr at RT.After that time the resulted mixture was poured, with stirring, into avessel containing 200 ml of ice/saturated sodium bicarbonate aqueoussolution. Organic compounds were extracted with 3×200 ml of ether. Theether fractions were combined and dried over sodium sulfate. Afterremoval of ether material was dissolved in minimum amount ofdichloromethane and purified by silica gel chromatography (100%dichloromethane as eluent). 1.92 gr (93% yield) of the desired productwere obtained.

16-Mercaptohexadecan-1-ol. Under inert atmosphere 365 mg of sodium metalsuspension (40% in mineral oil) were added dropwise to 20 ml of drymethanol at 0° C. After completion of addition the reaction mixture wasstirred for 10 min at RT followed by addition of 0.45 ml (6.30 mmole) ofthiolacetic acid. After additional 10 min of stirring 3 ml degassedmethanolic solution of 1.0 gr (3.11 mmole) of 16-bromohexadecan-1-olwere added. The resulted mixture was refluxed for 15 hrs, allowed tocool to RT and 20 ml of degassed 1.0 M NaOH aqueous solution wereinjected. The reaction completion required additional 3 hr of reflux.Resulted reaction mixture was cooled with ice bath and poured, withstirring, into a vessel containing 200 ml of ice water. This mixture wastitrated to pH=7 by 1.0 M HCl and extracted with 300 ml of ether. Theorganic layer was separated, washed with 3×150 ml of water, 150 ml ofsaturated NaCl aqueous solution and dried over sodium sulfate. Afterether removal material was dissolved in minimum amount ofdichloromethane and purified by silica gel chromatography (100%dichloromethane as eluent). 600 mg (70% yield) of the desired productwere obtained.

A clean gold covered microscope slide was incubated in a solutioncontaining 100 micromolar HS—(CH₂)₁₆—COOH in ethanol at room temperaturefor 4 hours. The electrode was then rinsed thoroughly with ethanol anddried. 20-30 microliters of wire-1 solution (1 micromolar in 1×SSCbuffer at pH 7.5) was applied to the electrode in a round droplet. Theelectrode was incubated at room temperature for 4 hours in a moistchamber to minimize evaporation. The wire-1 solution was then removedfrom the electrode and the electrode was immersed in 1×SSC bufferfollowed by 4 rinses with 1×SSC. The electrode was then stored at roomtemperature for up to 2 days in 1×SSC.

Alternatively, and preferably, either a “two-step” or “three-step”process is used. The “two-step” procedure is as follows. The wire-1compound, in water at ˜5-10 micromolar concentration, was exposed to aclean gold surface and incubated for ˜24 hrs. It was rinsed well withwater and then ethanol. The gold was then exposed to a solution of ˜100micromolar insulator thiol in ethanol for ˜12 hrs, and rinsed well.Hybridization was done with complement for over 3 hrs. Generally, thehybridization solution was warmed to 50° C., then cooled in order toenhance hybridization.

The “three-step” procedure uses the same concentrations and solvents asabove. The clean gold electrode was incubated in insulator solution for˜1 hr and rinsed. This procedure presumably results in an incompletemonolayer, which has areas of unreacted gold. The slide was thenincubated with wire-1 solution for over 24 hrs (generally, the longerthe better). This wire-1 still had the ethyl-pyridine protecting groupon it. The wire-1 solution was 5% NH4OH, 15% ethanol in water. Thisremoved the protecting group from the wire and allowed it to bind to thegold (an in situ deprotection). The slide was then incubated ininsulator again for ˜12 hrs, and hybridized as above.

In general, a variety of solvent can be used including water, ethanol,acetonitrile, buffer, mixtures etc. Also, the input of energy such asheat or sonication appears to speed up all of the deposition processes,although it may not be necessary. Also, it seems that longer incubationperiods for the wire-1 addition step, for example as long as a week, thebetter the results.

Hybridization efficiency was determined using ³²P complementary andnoncomplementary 15 mers corresponding to the wire-1 sequence. Theelectrodes were incubated with 50 microliters of each of the labellednon-complementary (herein “A5”) or complementary (herein “S5”) targetsequences applied over the entire electrode in 1×SSC as depicted inTable 1. The electrodes were then incubated for 1-2 hours at roomtemperature in a moist chamber, and rinsed as described above. Theamount of radiolabelled DNA was measured for each electrode in ascintillation counter, and the electrodes were dried and exposed toX-ray film for 4 hours.

TABLE 1 ³²P counts total ³²P counts hybridized to hybridized with: addedsurface A5, 20% specific activity, DNA 46,446   152 concentration 1 nM,1 hour incubation S5, 30% specific activity, DNA 39,166 10,484concentration 1 nM, 1 hour (27% incubation hybridized) A5, 14% specificactivity, DNA 182,020    172 concentration 5 nM, 2 hour incubation S5,20% specific activity, DNA 96,284 60,908 concentration 5 nM, 2 hour (63%incubation hybridized)

Example 7 Synthesis of Compositions Containing Ferrocene Linked to anElectrode

It has been shown in the literature that cyclic voltametry can be usedto determine the electron transfer rate of surface bound molecules.Surface bound molecules should show perfectly symmetric oxidation andreduction peaks if the scan speed of the voltammagram is sufficientlyslow. As the scan rate is increased, these peaks are split apart due tothe kinetics of electron transfer through the molecules. At a given scanspeed, a poorly conducting molecule should exhibit greater splittingthan a good conductor. As the speed is increased, the poor conductorwill be split even more.

Accordingly, to test the conductivity of the conductive polymer ascompared to a traditional insulator, two molecules were tested. Thesynthesis of ferrocene attached via a conductive oligomer to anelectrode (herein “wire-2”) was made as follows, as depicted in FIG. 7.

Synthesis of compound #11 was as follows. 2.33 gr (5.68 nmole) ofcompound #10 (made as described in Hsung et al., Organometallics14:4808-4815 (1995), incorporated by reference), 90 mg (0.47 mmole) ofCuI and 80 mg (0.11 mmole) of PdCl₂(PPh₃)₂ were dissolved in 100 ml ofpyrrolidine under inert atmosphere and heated for 20 hrs at 50° C. Allvolatile components were removed on high vacuum and resulted cruderesidue was dissolved in minimum amount of dichloromethane. The desiredcompound was purified by silica gel chromatography (50% ethylacetate+50% dichloromethane as eluent). 3.2 gr (90% yield) of the pureproduct were obtained.

Compound #12. To 200 mg (0.32 mmole) of suspension of MG#1 in 200 ml ofacetone (sonication was applied in order to get better results) 3 ml ofMeI were added and the reaction mixture was stirred for 20 hrs at RT.After that time volume of the resulted solution was reduced by rotovapevaporation to 50 ml and then 400 ml of n-hexane were added. Formedprecipitate was filtered out, washed with 3×200 ml of n-hexane and driedon high vacuum. Quantitative yield of the desired compound was obtained.

Compound #13. To 100 mg (0.13 mmole) of suspension of MG#2 in 200 ml ofacetone (sonication was applied in order to get better results) 10 ml oftriethyl amine were added and the reaction mixture was stirred for 20hrs at RT. After that time volume of the resulted solution was reducedby rotovap evaporation to 50 ml and then 400 ml of n-hexane were added.Formed precipitate was filtered out, washed with 3×200 ml of n-hexaneand dried on high vacuum. The desired compound was extracted from thisprecipitate with 3×50 ml of THF. Evaporation of the THF fractions gave35 mg (52%) of the compound #13. This was then added to a gold electrodeas known in the art.

HS—(CH2)15NHCO-Fc (herein “insulator-1”) was made as described in Wardet al., Anal. Chem. 66:3164-3172 (1994), hereby incorporated byreference (note: the FIG. 1 data has been shown to be incorrect,although the synthesis of the molecule is correct).

Monolayers of each were made as follows. Insulator: Gold coveredmicroscope slides were immersed in a mixture of insulator-1 andHS—(CH2)15-OH (insulator-2) in neat ethanol. Insulator-2 molecule isadded to the mixture to prevent the local concentration of ferrocene atany position from being too high, resulting in interactions between theferrocene molecules. The final solution was 0.1 mM insulator-1 and 0.9mM insulator-2. The mixture was sonicated and heated (60-80° C.) for1-10 hours. The electrodes were rinsed thoroughly with ethanol, waterand ethanol. The electrodes were immersed in a 1 mM thiol solution inneat ethanol and let stand at room temperature for 2-60 hours. Theelectrodes were then rinsed again. This procedure resulted in 1-10%coverage of insulator-1 as compared to calculated values of close packedferrocene molecules on a surface. More or less coverage could easily beobtained by altering the mixture concentration and/or incubation times.

Wires: The same procedure was followed as above, except that the secondstep coating required between 10 and 60 hours, with approximately 24hours being preferable. This resulted in lower coverages, with between0.1 and 3% occurring.

Cyclic voltametry was run at 3 scan speeds for each compound: 1 V/sec,10 V/ec, and 50 V/sec. Even at 1 V/sec, significant splitting occurswith insulator-1, with roughly 50 mV splitting occuring. At higherspeeds, the splitting increases. With wire-2, however, perfectlysymmetrical peaks are observed at the lower speeds, with only slightsplitting occurring at 50 V/sec.

It should be noted that despite a significant difference in electrontransfer rate, electron transfer does still occur even in poorlyconducting oligomers such as (CH₂)₁₅, traditionally called “insulators”.Thus the terms “conductive oligomer” and “insulator” are somewhatrelative.

Example 8 Synthesis and Analysis of Nucleic Acid with Both a ConductiveOligomer and a Second Electron Transfer Moiety

The following nucleic acid composition was made using the techniquesabove: 5′-ACCATGGAC[UBF]CAGCU(SEQ ID NO:2)-conductive polymer (Structure5 type, as outlined above) herein “wire-3”, with UBF made as describedabove. Thus, the second electron transfer moiety, ferrocene, is on thesixth base from the conductive oligomer.

Mixed monolayers of wire-3 and insulator-2 were constructed using thetechniques outlined above. The compositions were analyzed in 0.2 MNaClO₄ in water using cyclic voltametry (CV) and square wave voltametry(SW), in the absence (i.e. single stranded) and presence (i.e. doublestranded) of complementary target sequence.

The results of SW show the absence of a peak prior to hybridization,i.e. in the absence of double stranded nucleic acid. In the presence ofthe complementary target sequence, a peak at ˜240 mV, corresponding toferrocene, was seen.

A mediator as described herein was also used. 6 mM ferricyanide(Fe(CN)₆) was added to the solution. Ferricyanide should produce a peakat 170 mV in a SW experiment. However, no peak at 170 mV was observed,but the peak at 240 mV was greatly enhanced as compared to the absenceof ferricyanide.

Alternatively, CV was done. No peaks were observed in the absence oftarget sequence. Once again, the chip was incubated with perfectlycomplimentary nucleic acid in order to hybridize the surface nucleicacid. Again, the chip was scanned under the same conditions. Anincreased signal was observed. Finally, the chip was soaked in buffer at70° C. in order to melt the compliment off the surface. Previousexperiments with radioactive probes have shown that 15-mers hybridizedon a very similar surface melted at approximately 45° C. Repeating thescan after the heat treatment shows a reduced signal, as in the firstscan prior to hybridization.

Example 9 AC Detection Methods

Electrodes containing four different compositions of the invention weremade and used in AC detection methods. In general, all the electrodeswere made by mixing a ratio of insulator-2 with the sample as isgenerally outlined above.

Sample 1, labeled herein as “Fc-alkane”, contained a mixed monolayer ofinsulator-2 and insulator-1. Sample 2, labeled herein as“Fc-amido-alkane”, contained a mixed monolayer of insulator-2 and aderivative of insulator-1 which has an amido attachment of the ferroceneto the alkane. Sample 3, labeled herein as “Fc-wire”, contained a mixedmonolayer of insulator-2 and wire-2. Sample 4 was the same as Sample 3,with the exception that a new in situ deprotection step was used,described below. Sample 5, labeled herein as “ssDNA”, contained a mixedmonolayer of insulator-2 and wire-3.

Sample 6, labeled herein as “dsDNA”, contained a mixed monolayer ofinsulator-2 and wire-3, wherein the complement of wire-3 was hybridizedto form a double stranded wire-3. Sample 7 was a solution of ferrocenein solution. As is shown herein, the rate of electron transfer, fromfast to slower, is as follows: Sample 3>Sample 5>Sample 1>Sample 4.Generally, Sample 1 models ssDNA, and Sample 3 models dsDNA.

The experiments were run as follows. A DC offset voltage between theworking (sample) electrode and the reference electrode was swept throughthe electrochemical potential of the ferrocene, typically from 0 to 500mV. On top of the DC offset, an AC signal of variable amplitude andfrequency was applied. The AC current at the excitation frequency wasplotted versus the DC offset.

FIG. 8 depicts an experiment with Sample 1, at 200 mV AC amplitude andfrequencies of 1, 5 and 100 Hz. Sample 1 responds at all threefrequencies, and higher currents result from higher frequencies, whichis simply a result of more electrons per second being donated by theferrocene at higher frequencies. The faster the rate, the higher thefrequency response, and the better the detection limit. FIG. 9 showsoverlaid AC voltammograms of an electrode coated with Sample 3. Fourexcitation frequencies were applied: 10 Hz, 100 Hz, 1 kHz, 10 kHz, allat 25 mV overpotential. FIG. 10, shows the frequency response of thesystem by measuring the peak currents. Sample 3 response to increasingfrequencies through 10 kHz (the detector system limit), while Sample 1lose its responses at between 20 and 200 Hz. Thus, to discriminatebetween Sample 1 and Sample 3, one would only have to analyze it at 1 Hzand 1000 Hz and compare the responses. This should be similar to thedsDNA and ssDNA system. FIG. 11 shows Sample 5 and Sample 6, plotted asa function of normalized current (with the highest current being 1 forboth cases; the actual current of dsDNA is much higher than that ofssDNA, so the graph was normalized to show both). The lines are modeledRC circuits, as described above, and not a fit to the data. At 1 Hz,both ssDNA and dsDNA respond; at 200 Hz, the ssDNA signal is gone. FIG.12 shows that increasing the overpotential will increase the outputsignal. FIGS. 13A and 13B show that the overpotential and frequency canbe tuned to increase the selectivity and sensitivity. For example, a lowoverpotential and high frequence can be used to minimize the slowerspecies (Sample 1 or Sample 5). Then the overpotential can be increasedto induce a response in the slower species for calibration andquantification.

FIG. 14 shows that the ferrocene added to the solution (Sample 7) has afrequency response related to diffusion that is easily distinguishablefrom the frequency response of attached ferrocene. This indicates thatby varying frequency, signals from bound molecules, particular fastbound molecules such as dsDNA, can be easily distinguished from anysignal generated by contaminating redox molecules in the sample.

FIGS. 15A and 15B shows the phase shift that results with differentsamples. FIG. 15A shows the model compounds, and 15B shows data withdsDNA and ssDNA. While at this frequency, the phase shift is not large,a frequency can always be found that results in a 90° shift in thephase.

Example 10 Synthesis of Conductive Oligomers Attached Via a Base

Representative syntheses are depicted in FIGS. 18 and 19. When usingpalladium coupling chemistry, it appears that protecting groups arerequired on the base, in order to prevent significant dimerization ofconductive oligomers instead of coupling to the iodinated base. Inaddition, changing the components of the palladium reaction may bedesirable also. Also, for longer conductive oligomers, R groups arepreferred to increase solubility.

Example 11 The Use of Trimethylsilylethyl Protecting Groups

The use of an alternate protecting group for protection of the sulfuratom prior to attachment to the gold surface was explored.

To 0.5 gm of molecular sieve (3 Å) was added 3 ml of dry THF and 2.5 mlof 1.0 tetrabutylammonium fluoride. After stirring for 20 minutes, 100mg of compound #1 was added under Argon. The reaction mixture wasstirred for 1 hour and poured into 100 ml of 5% citric acid solution andthe aqueous solution was shaken well and extracted twice with either(2×100 ml). The combined ether solution was dried over Na₂SO₄ andconcentrated. The residue was purified by column chromatography using10% CH₂Cl₂/Hexane as eluent. The purified product was analyzed by ¹HNMRwhich should 50% of compound #2 and 50% of the corresponding disulfide.

The use of this protecting group in synthesizing base-attachedconductive oligomers is depicted in FIGS. 20 and 21.

1. An apparatus for the detection of target nucleic acids in a testsample, comprising: a) a test chamber comprising a first and a secondelectrode, wherein said first electrode comprises: i. a single strandednucleic acid covalently attached to said electrode via an insulator,wherein said insulator is an alkyl oligomer wherein each monomer of saidalkyl oligomer is independently selected from the group consisting of:—(CH₂)_(n)—, —(CRH)_(n)—, —(CR2)_(n)—, ethylene glycol and ethyleneglycol derivatives using other heteroatoms in place of oxygen; wherein nis from 1 to 16; and wherein R is selected from the group consisting ofhydrogen, alkyl, alcohol, aromatic amino, amido, nitro, ethers, esters,aldehydes, sulfonyl, silicon moieties, halogens, sulfur containingmoieties, phosphorous containing moieties, and ii. a passivation agentmonolayer; and b) an AC/DC voltage source electrically connected to saidfirst and second electrodes.
 2. An apparatus accordingly to claim 1further comprising, c) a second nucleic acid covalently attached to anelectron transfer moiety.
 3. An apparatus for the detection of targetnucleic acids in a test sample, comprising: a) a test chamber comprisinga first and a second electrode, wherein said first electrode comprises:i. a covalently attached single stranded nucleic acid attached to saidfirst electrode via an insulator, wherein said insulator is an alkyloligomer wherein each monomer of said alkyl oligomer is independentlyselected from the group consisting of: —(CH₂)_(n)—, —(CRH)_(n)—,ethylene glycol and ethylene glycol derivatives using other heteroatomsin place of oxygen; wherein n is from 1 to 16; and wherein R is selectedfrom the group consisting of hydrogen, alkyl alcohol, aromatic amino,amido, nitro, ethers, esters, aldehydes, sulfonyl, silicon moieties,halogens, sulfur containing moieties, phosphorous containing moieties;ii. a passivation agent monolayer; and iii. a covalently attached firstelectron transfer moiety; and b) an AC/DC voltage source electricallyconnected to said test chamber.
 4. An apparatus for the detection oftarget nucleic acids in a test sample, comprising: a. a test chambercomprising a first and a second electrode, wherein said first electrodecomprises: i. a covalently attached first single stranded nucleic acidattached to said first electrode via an insulator, wherein saidinsulator is an alkyl oligomer wherein each monomer of said alkyloligomer is independently selected from the group consisting of:—(CH₂)_(n)—, —(CRH)_(n)—, —(CR2)_(n)—, ethylene glycol and ethyleneglycol derivatives using other heteroatoms in place of oxygen; wherein nis from 1 to 16; and wherein R is selected from the group consisting ofhydrogen, alkyl, alcohol, aromatic amino, amido, nitro, ethers, esters,aldehydes, sulfonyl, silicon moieties, halogens, sulfur containingmoieties, phosphorous containing moieties; and ii. a passivation agentmonolayer; and b) a second nucleic acid covalently attached to anelectron transfer moiety; and c) an AC/DC voltage source electricallyconnected to said test chamber.
 5. An apparatus according to claim 1, 3or 4, further comprising: a processor coupled to said electrodes.
 6. Anapparatus according to claim 1, 3 or 4, wherein said AC/DC voltagesource is capable of delivering frequencies from between about 1 Hz toabout 100 kHz.
 7. An apparatus according to claim 1, 3 or 4 wherein saidpassivation agent monolayer comprises conductive oligomers.
 8. Anapparatus according to claim 1, 3 or 4 wherein said passivation agentmonolayer comprises insulators.
 9. An apparatus according to claims 1, 3or 4 wherein said passivation agent monolayer comprises alkyl chains.10. An apparatus according claim 9 wherein said alkyl chains have theformula C_(n)H_(x), where n is 1 to 30, and x is 2(n).
 11. An apparatusaccording to claims 1, 3 or 4 wherein said passivation agent monolayercomprises terminal groups chosen from the group consisting of—(CH₂)_(n)—, —(CRH)_(n)—, —(CR2)_(n)—, ethylene glycol and ethyleneglycol derivatives using other heteroatoms in place of oxygen; wherein nis from 1 to 16, and wherein R is selected from the group consisting ofhydrogen, alkyl, alcohol, aromatic amino, amido, nitro, ethers, esters,aldehydes, sulfonyl, silicon moieties, halogens, sulfur containingmoieties, phosphorous containing moieties, and ethylene glycol moieties.12. An apparatus according to claims 1, 3 or 4 wherein said passivationagent monolayer comprises both conductive oligomers and insulators. 13.An apparatus for the detection of target nucleic acids in a test sample,comprising: a) a test chamber comprising an array of electrodes, eachelectrode comprising i. a covalently attached single stranded nucleicacid, attached to said electrodes via an insulator, wherein saidinsulator is an alkyl oligomer wherein each monomer of said alkyloligomer is independently selected from the group consisting of:—(CH₂)_(n)—, —(CRH)_(n)—, —(CR2)_(n)—, ethylene glycol and ethyleneglycol derivatives using other heteroatoms in place of oxygen; wherein nis from 1 to 16; and wherein R is selected from the group consisting ofhydrogen, alkyl, alcohol, aromatic amino, amido, nitro, ethers, esters,aldehydes, sulfonyl, silicon moieties, halogens, sulfur containingmoieties, phosphorous containing moieties; and ii. a passivation agentmonolayer; and b) an AC/DC voltage source electrically connected to saidtest chamber.
 14. An apparatus according to claim 13 wherein saidpassivation agent monolayer comprises conductive oligomers.
 15. Anapparatus according to claim 13 wherein said passivation agent monolayercomprises insulators.